3.3 The Cell Wall and Outer Layers

How do bacteria and archaea protect their cell membrane? For most species, the cell envelope includes at least one structural supporting layer, like an external skeleton, located outside the cell membrane. The most common structural support is the cell wall (see Fig. 3.1). Many species possess additional coverings, such as an outer membrane or an S-layer. Nevertheless, a few prokaryotes, such as the mycoplasmas, have a cell membrane with no outer layers, depending on host fluids for osmotic balance. Some archaea with only a cell membrane grow in extreme acid (pH zero); how they survive is unknown.

The Cell Wall Is a Single Molecule

The bacterial cell wall, also known as the sacculus, consists of a single interlinked molecule that envelops the cell. The sacculus has been isolated from E. coli and visualized by TEM; in Figure 3.14A, the isolated sacculus appears flattened on the sample grid like a deflated balloon. Its geometrical structure encloses maximal volume with minimal surface area. The sacculus—unlike the membrane—is a single-molecule cage-like structure, highly porous to ions and organic molecules. The mesh grows by strand insertion and elongation in arcs around the cell. The cage-like form is not rigid; it is more like a flexible mesh bag with unbreakable joints. Turgor pressure within the enclosed cytoplasm fills out the cell’s shape, such as a rod or a spherical coccus.

FIGURE 3.14 The peptidoglycan sacculus and peptidoglycan cross-bridge formation. A. Isolated sacculus from Escherichia coli (TEM). B. A disaccharide unit of glycan has an attached peptide of four to six amino acids.

Peptidoglycan structure. Most bacterial cell walls are composed of peptidoglycan, a polymer of peptide-linked chains of amino sugars. Peptidoglycan is synonymous with murein (“wall molecule”). The molecule consists of parallel polymers of disaccharides called glycan chains cross-linked with peptides of four amino acids (Fig. 3.14B). Peptidoglycan is unique to bacteria, although some archaea build analogous structures whose overall physical nature is similar. (Archaeal cell walls are presented in Chapter 19.)

The long chains of peptidoglycan consist of repeating units of a disaccharide composed of N-acetylglucosamine (an amino sugar derivative) and N-acetylmuramic acid (glucosamine plus a lactic acid group) (Fig. 3.14B). The lactate group of muramic acid forms an amide link with the amino terminus of a short peptide containing four to six amino acid residues. The peptide extension can form cross-bridges connecting parallel strands of glycan.

The peptide contains two amino acids in the unusual D mirror form: D-glutamic acid and D-alanine. The third amino acid, m-diaminopimelic acid, has an extra amine group, which forms an amide link to a cross-bridged peptide. The amide link forms with the fourth amino acid of the adjacent peptide, D-alanine (1) (Fig. 3.14B). The cross-bridge forms by removal of a second D-alanine (2) at the end of the chain. The cross-linked peptides of neighboring glycan strands form the cage of the sacculus.

Note: Amino acids have two forms that are mirror opposites, D and L, of which only the L form is incorporated by ribosomes into protein. The D-form amino acids, however, are used by microbes for many nonprotein structural molecules.

The details of peptidoglycan structure vary among bacterial species. Some Gram-positive species, such as Staphylococcus aureus (a cause of toxic shock syndrome), have peptides linked by bridges of pentaglycine instead of the D-alanine link to m-diaminopimelic acid. In Gram-negative species, the m-diaminopimelic acid is linked to the outer membrane, as discussed shortly.

Peptidoglycan synthesis as a target for antibiotics. Synthesis of peptidoglycan requires many genes encoding enzymes to make the special sugars, build the peptides, and seal the cross-bridges. Many of these enzymes bind the antibiotic penicillin and are thus known as penicillin-binding proteins. Because peptidoglycan is unique to bacteria, enzymes of peptidoglycan biosynthesis make excellent targets for new antibiotics (see Fig. 3.14B). For example, the transpeptidase that cross-links the peptides is the target of penicillin. Vancomycin, a major defense against Clostridium difficile and drug-resistant staphylococci, prevents cross-bridge formation by binding the terminal D-Ala-D-Ala dipeptide. Vancomycin binding prevents release of the terminal D-alanine.

Unfortunately, the widespread use of such antibiotics selects for evolution of resistant strains. One of the most common agents of resistance is the enzyme beta-lactamase, which cleaves the lactam ring of penicillin, rendering it ineffective as an inhibitor of transpeptidase. In a different mechanism, strains resistant to vancomycin contain an altered enzyme that adds lactic acid to the end of the branch peptides in place of the terminal D-alanine. The altered peptide is no longer blocked by vancomycin. As new forms of drug resistance emerge, researchers continue to seek new antibiotics that target cell wall formation (discussed in Chapter 27).

How does peptidoglycan grow, overall, throughout the elongating cell? Interestingly, different kinds of bacteria have evolved to organize their cell wall growth differently. Yves Brun and his student Erkin Kuru, at Indiana University, devised an ingenious way to reveal the growth pattern (Fig. 3.15). Kuru designed D-amino acids with fluorophores attached that the growing cell wall incorporates. Because these are D-amino acids and not L-amino acids, ribosome-directed translation does not use them. Thus, the fluorophores label only cell wall, not proteins. Kuru found that bacteria such as E. coli and Bacillus subtilis synthesize cell wall in zones dispersed throughout the cell. Gram-positive cocci, however, such as Streptococcus and Staphylococcus, synthesize cell wall only at the midpoint (or septum), where the cell is about to fission (discussed in the next section). Still other bacteria, such as the actinomycete Streptomyces, form new cell wall only at the poles.

FIGURE 3.15 Peptidoglycan growth in different species. A. Different species of bacteria synthesize new peptidoglycan in dispersed zones, at the septum only, or at the poles. Fluorescent D-amino acids are added for short periods (pulse labeling) to reveal the growth zones. B. Erkin Kuru, in the laboratory of Yves Brun at Indiana University, devised the fluorescent D-amino acids to probe cell wall growth.

Cell Envelope of Bacteria

Most bacteria have additional envelope layers that provide structural support and protection from predators and host defenses (Fig. 3.16). Additional molecules are attached to the cell wall and cell membrane, and some thread through the layers. Here we present the envelope composition of three major kinds of bacteria, two of which (Firmicutes and Proteobacteria) are distinguished by the Gram stain. The third, Mycobacteria, is distinguished by the acid-fast stain (discussed in Chapter 2).

  • Firmicutes (Gram-positive) have a thick cell wall with 3–20 layers of peptidoglycan, interpenetrated by teichoic acids. The phylum Firmicutes consists of Gram-positive species such as Bacillus thuringiensis and Streptococcus pyogenes, the cause of strep throat.
  • Proteobacteria (Gram-negative) have a thin cell wall with one or two layers of peptidoglycan, enclosed by an outer membrane. The phylum Proteobacteria consists of Gram-negative species such as Escherichia coli and nitrogen-fixing Sinorhizobium meliloti.
  • Mycobacteria of the phylum Actinomycetes have a complex multilayered envelope that includes defensive structures such as mycolic acids. Examples include Mycobacterium tuberculosis, the cause of tuberculosis, and M. leprae, the cause of leprosy.

FIGURE 3.16 Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria). A. Firmicutes (Gram-positive) cells have a thick cell wall with multiple layers of peptidoglycan, threaded by teichoic acids. A inset: Gram-positive envelope of Bacillus subtilis (TEM). B. Proteobacteria (Gram-negative) cells have a single layer of peptidoglycan covered by an outer membrane; the cell membrane is called the inner membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).

Note that other important kinds of bacteria, such as cyanobacteria and spirochetes, have very different envelopes. These different envelope structures may stain Gram-positive, Gram-negative, or variable (discussed in Chapter 18). Archaea have yet other diverse kinds of envelopes that cannot be distinguished by Gram stain (see Chapter 19).

Firmicute Cell Envelope—Gram-Positive

FIGURE 3.17 Teichoic acids. Teichoic acids in the Gram-positive cell wall consist of glycerol or ribitol phosphodiester chains.

A section of a Firmicute cell envelope (Gram-positive) is shown in Figure 3.16. The multiple layers of peptidoglycan are reinforced by teichoic acids threaded through its multiple layers (Fig. 3.17). Teichoic acids are chains of phosphodiester-linked glycerol or ribitol, with sugars or amino acids linked to the middle −OH groups. The negatively charged cross-threads of teichoic acids, as well as the overall thickness of the Gram-positive cell wall, help retain the Gram stain.

How does the cell wall attach extracellular structures? Gram-positive bacteria have a type of enzyme called “sortase” that forms a peptide bond from a cell wall cross-bridge to a protein extending from the cell. Proteins attached by sortases can help the cell acquire nutrients, or help the cell adhere to a substrate. Sortases are now used in the protein engineering industry.

S-layer. Free-living bacteria and archaea often possess a tough surface layer called the S-layer. Figure 3.18 shows the S-layer of Lysinibacillus sphaericus, a Gram-positive bacterium found on the surface of beets and carrots. The S-layer is a crystalline sheet of thick subunits consisting of protein or glycoprotein (proteins with attached sugars). Each subunit contains a pore large enough to admit a wide range of molecules (Fig. 3.18). As modeled by cryo-EM tomography (see Chapter 2), the S-layer proteins are arranged in a highly ordered array, either hexagonally or tetragonally. The S-layer is rigid, but it also flexes and allows substances to pass through it in either direction. S-layers help pathogens such as Bacillus anthracis bind and attack host cells. An S-layer contributes to biofilm formation (the periodontal bacterium Tannerella forsythia) and swimming (the aquatic cyanobacterium Synechococcus species).

FIGURE 3.18 S-layer of Lysinibacillus bacteria. A. Lysinibacillus imaged by atomic force microscopy (AFM). B. S-layer surface imaged by AFM. C. Computational model of S-layer based on cryo-EM.

The functions of the S-layer are hard to study in the laboratory because the S-layer is often lost by bacteria after repeated subculturing. Traits commonly diminish in the absence of selective pressure for genes encoding them—a process called reductive evolution (discussed in Chapter 17). For example, the mycoplasmas are close relatives of Gram-positive bacteria that have permanently lost their cell walls, as well as the S-layer. Mycoplasmas have no need for cell walls, because they are parasites living in host environments, such as the human lung, where they are protected from osmotic shock.

Capsule. Another common extracellular structure is the capsule, a slippery layer of loosely bound polysaccharides. The capsule of pathogens such as Staphylococcus aureus can prevent phagocytosis by white blood cells, thereby enabling the pathogen to persist in the blood.

Proteobacterial Cell Envelope—Gram-Negative

A cell envelope of Proteobacteria (Gram-negative) is shown in Figures 3.16B and 3.19. The envelope includes one or two layers of peptidoglycan covered by an outer membrane. The Gram-negative outer membrane confers defensive abilities and toxigenic properties on many pathogens, such as Salmonella species and enterohemorrhagic E. coli (strains that cause hemorrhaging of the colon). Between the outer and inner (cell) membranes, the aqueous compartment (containing the cell wall) is called the periplasm.

FIGURE 3.19 Gram-negative cell envelope. A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall. B. Lack of murein lipoprotein in mutant Salmonella causes the outer membrane to balloon out (arrow), when the cell tries to divide (TEM).

Lipoprotein and lipopolysaccharide (LPS). The inward-facing leaflet of the outer membrane has a phospholipid composition similar to that of the cell membrane (which in Gram-negative species is called the inner membrane or inner cell membrane). The outer membrane’s inward-facing leaflet includes lipoproteins that connect the outer membrane to the peptide bridges of the cell wall. The major lipoprotein is called murein lipoprotein, also known as Braun lipoprotein (Fig. 3.19A). Murein lipoprotein consists of a protein with an N-terminal cysteine attached to three fatty acid side chains. The side chains are inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of peptidoglycan (murein). What happens to a mutant cell that fails to make murein lipoprotein? As the cell grows and divides, it fails to attach its outer membrane to the growing cell wall, causing the outer membrane to balloon out in the region where the daughter cells separate (Fig. 3.19B).

FIGURE 3.20 Lipopolysaccharide (LPS). A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to six fatty acids. B. Repeating polysaccharide units of O antigen extend from lipid A.

The outward-facing leaflet of the outer membrane has very different lipids from the inner leaflet. The main outward-facing phospholipids are called lipopolysaccharide (LPS) (Fig. 3.20). LPS is of crucial medical importance because it acts as an endotoxin. An endotoxin is a cell component that is harmless as long as the pathogen remains intact; but when released by a lysed cell, endotoxin overstimulates host defenses, inducing potentially lethal endotoxic shock. Thus, antibiotic treatment of an LPS-containing pathogen can kill the cells but can also lead to death of the patient.

The membrane-embedded anchor of LPS is lipid A, a molecule shaped like a six-legged giraffe (Fig. 3.20A). The lipid A moiety is the endotoxic part of LPS. The molecule’s six fatty acid “legs” have shorter chains than those of the inner cell membrane, and two pairs are branched. The fatty acids have ester or amide links to the “body,” a dimer of glucosamine (an amino sugar also found in peptidoglycan). Analogous to the glycerol of glyceride phospholipids, each glucosamine has a phosphoryl group whose negative charge interacts with water. One glucosamine extends the long “neck” of the core polysaccharide, a sugar chain that reaches far outside the cell (Fig. 3.20B). The core polysaccharide consists of five to ten sugars with side chains such as phosphoethanolamine. It extends to an O antigen or O polysaccharide, a chain of as many as 200 sugars. The O polysaccharide may be longer than the cell itself. These chains of sugars form a layer that helps bacteria resist phagocytosis by white blood cells. The combination of sugar units in the O antigen varies greatly among different strains and species of Gram-negative bacteria.

FIGURE 3.21 OmpF porin transports ampicillin. This model of the OmpF trimer is based on X-ray crystallography. (PDB code: 2OMF) Within each tubular porin monomer, charged amino acid residues contact ampicillin. Source: Modified from Ekaterina M. Nestorovich et al. 2002. PNAS 99:9789–9794.

Outer membrane proteins. The outer membrane contains unique proteins not found in the inner membrane. Outer membranes contain a class of transporters called porins that permit the entry of nutrients such as sugars and peptides (Fig. 3.21). Outer membrane porins such as OmpF have a distinctive cylinder of beta sheet conformation (reviewed in eAppendix 1), also known as a beta barrel. A typical outer membrane porin exists as a trimer of beta barrels, each of which acts as a pore for nutrients.

Outer membrane porins have limited specificity, allowing passive uptake of various molecules—including antibiotics such as ampicillin. Ampicillin is a form of penicillin, which must get through the outer membrane to access the cell wall in order to block the formation of peptide cross-bridges. Ampicillin contains two charged groups and is thus unlikely to diffuse through a lipid bilayer. But the molecule crosses the E. coli outer membrane by passing through OmpF, where its charged groups are attracted to charged amino acid residues extending inside the porin (Fig. 3.21). Ampicillin’s positively charged amine group is attracted to the carboxylate of glutamate-117, and its negatively charged carboxylate is attracted to the arginine amines. Experiments demonstrating ampicillin passage through OmpF are described in eTopic 3.2.

If porins can admit dangerous molecules as well as nutrients, should a cell make porins or not? In fact, cells express different outer membrane porins under different environmental conditions. In a dilute environment, cells express porins of large pore size, maximizing the uptake of nutrients. In a rich environment—for example, within a host—cells down-regulate the expression of large porins and express porins of smaller pore size, selecting only smaller nutrients and avoiding the uptake of toxins. For example, the porin regulation system of Gram-negative bacteria enables them to grow in the colon containing bile salts—a hostile environment for Gram-positive bacteria, which lack an outer membrane.

Periplasm. The outer membrane is porous to most ions and many small organic molecules, but it prevents the passage of proteins and other macromolecules. Thus, the region between the inner and outer membranes of Gram-negative cells, including the cell wall, defines a separate membrane-enclosed compartment of the cell known as the periplasm (see Fig. 3.19). The periplasm contains specific enzymes and nutrient transporters not found within the cytoplasm, such as periplasmic transporters for sugars, amino acids, or other nutrients. Periplasmic proteins are subjected to fluctuations in pH and salt concentration, because the outer membrane is porous to ions. Some periplasmic proteins help refold proteins unfolded by oxidizing agents or by acidification.

Overall, the outer membrane, periplasm, inner membrane, and cytoplasm define four different cellular compartments within a Gram-negative cell: two membrane-soluble compartments (outer and inner membrane), and two aqueous compartments (periplasm and cytoplasm). Each type of cellular protein is typically found in only one of these locations. For example, the proton-translocating ATP synthase is found only in the inner membrane fractions, whereas sugar-accepting porins are only in the outer membrane.

S-layer and capsule. Some Gram-negative bacteria also form an S-layer and/or a capsule exterior to the outer membrane. For example, an S-layer is found in Caulobacter, an aquatic proteobacterium; and in species of the genus Campylobacter, which cause diarrheal disease and systemic infections of immunocompromised patients. A capsule is found in virulent strains of Haemophilus influenzae, which was the leading cause of childhood meningitis before development of the Hib vaccine (see Chapter 24).

Mycobacterial Cell Envelope

FIGURE 3.22 Mycobacterial envelope structure. A complex cell wall includes a peptidoglycan layer linked to a chain of galactose polymer (galactan) and arabinose polymer (arabinan). Arabinan forms ester links to mycolic acids, which form an outer bilayer with phenolic glycolipids.

Exceptionally complex cell envelopes are found in actinomycetes, a large and diverse family of soil bacteria that produce antibiotics and other industrially useful products (discussed in Chapter 18). The most complex envelopes known are those of actinomycete-related bacteria, the mycobacteria. Mycobacteria include the famous pathogens Mycobacterium tuberculosis (the cause of tuberculosis) and Mycobacterium leprae (the cause of leprosy). The mycobacterial envelope includes features of both Gram-positive and Gram-negative cells, as well as structures unique to mycobacteria (Fig. 3.22).

The thick mycobacterial cell wall offers physical protection comparable to the cell wall of Gram-positive bacteria. In mycobacteria, the peptidoglycan is linked to chains of galactose, called galactans. The galactans are attached to arabinans, polymers of the five-carbon sugar arabinose. The arabinan-galactan polymers are known as arabinogalactans. Arabinogalactan biosynthesis is inhibited by two major classes of anti-tuberculosis drugs: ethambutol and the benzothiazinones.

The ends of the arabinan chains form ester links to mycolic acids (uncharged mycolates). Mycolic acids provide the basis for acid-fast staining, in which cells retain the dye carbolfuchsin, an important diagnostic test for mycobacteria and actinomycetes (described in Chapter 28). Mycolic acids contain a hydroxy acid backbone with two hydrocarbon chains—one comparable in length to typical membrane lipids (about 20 carbons), the other about threefold longer. The long chain includes ketones, methoxyl groups, and cyclopropane rings. Hundreds of different forms are known. The mycolic acids form a bilayer interleaved with sugar mycolates—a kind of outer membrane, analogous to the Gram-negative outer membrane. This mycolate layer even contains porins homologous to Gram-negative beta barrel porins such as OmpA. Other mycolate-embedded proteins include virulence factors such as fibronectin-binding protein (Fbp). Fbp enhances the ability of M. tuberculosis to invade macrophages.

The outer ends of the sugar mycolates are interleaved with phenolic glycolipids, which include phenol groups linked to sugar chains. The extreme hydrophobicity of the phenol derivatives generates a waxy surface that prevents phagocytosis by macrophages. Overall, the thick, waxy envelope excludes many antibiotics and offers exceptional protection from host defenses, enabling the pathogens of tuberculosis and leprosy to colonize their hosts over long periods. However, the thick envelope also retards uptake of nutrients. As a result, M. tuberculosis and M. leprae grow extremely slowly and are a challenge to culture in the laboratory.

Bacterial Cytoskeleton

In eukaryotes, cell shape has long been known to be maintained by a cytoskeleton of protein microtubules and filaments (reviewed in eAppendix 2). But what determines the shape of bacteria? We saw earlier that bacterial shape is in part maintained by the cell wall and the resulting turgor pressure. But research over the past decade shows that bacteria also possess protein cytoskeletal components—and remarkably, some of them are homologous to eukaryotic cytoskeletal proteins. For example, the cell division protein FtsZ is a homolog of the eukaryotic protein tubulin (the subunit of eukaryotic microtubules). Another bacterial cytoskeletal protein, MreB, is a homolog of the eukaryotic microfilament protein actin.

FIGURE 3.23 Cytoskeletal mutants. Wild-type cells compared with mutants in Bacillus subtilis (A; fluorescence microscopy) and Caulobacter crescentus (B; DIC microscopy).

The bacterial cytoskeletal proteins are revealed by gene defects that drastically alter the cell shape. For example, Figure 3.23A compares wild-type cells of Bacillus subtilis with cells containing mutations in three mreB homologs (mreB, mreI, and mreBH). The wild-type cells have a defined rod shape, whereas the mutant shape is round and undefined. The mutant lacks the MreB complex that regulates peptidoglycan synthesis and thereby defines the rod-shaped cell. Another example of a shape-altering mutation affects the comma-shaped cell of Caulobacter crescentus (Fig. 3.23B). A mutation in gene creS results in cells that are straight instead of curved. The creS gene expresses the cytoskeletal protein CreS (crescentin).

How do the various cytoskeletal proteins work together to generate the overall shape of a bacterial cell? The functions of cytoskeletal proteins are probed by fluorescent protein fusions (Fig. 3.24). In both spherical bacteria (cocci) and rod-shaped bacilli, the FtsZ complex Z-ring determines the cell diameter. Elongation of a rod-shaped cell requires polymerization of a second cytoskeletal protein, MreB (Fig. 3.24B). MreB localizes in arc-shaped patches, just beneath the cell membrane of the rod-shaped cell. Some models propose a helical coil of MreB around the entire cell, but more recent data indicate arcs of MreB driven by elongating strands of cell wall. If the rod shape is curved (called a vibrio, or crescent shape), the third cytoskeletal protein, crescentin, polymerizes along the inner curve of the crescent (Fig. 3.24C). The cell’s outer curve is visualized by a membrane-specific fluorophore. These cytoskeletal proteins, and their variants that have evolved in other species, work together within cells to generate the shapes of bacteria.

FIGURE 3.24 Shape-determining proteins. A. Cell diameter is maintained by FtsZ polymerization to form the Z-ring. B. Elongation of a rod-shaped cell requires Mre proteins. MreB polymerizes around an E. coli cell (MreB-YFP fluorescence) along with a Z-ring of FtsZ (fluorescent anti-FtsZ antibody). C. Crescent-shaped cells possess a third shape-determining protein, CreS (crescentin), which polymerizes along the inner curve of the crescent. Crescentin protein fused to green fluorescent protein (CreS-GFP) localizes to the inner curve of Caulobacter crescentus. Membrane-specific stain FM4-64 (red fluorescence) localizes to the membrane around the cell.

To Summarize
  • The cell wall maintains turgor pressure. The cell wall is porous, but its network of covalent bonds generates turgor pressure that protects the cell from osmotic shock.
  • The Gram-positive cell envelope has multiple layers of peptidoglycan, threaded by teichoic acids.
  • The S-layer of proteins is highly porous but can prevent phagocytosis and protect cells in extreme environments. In archaea, the S-layer serves the structural function of a cell wall.
  • The capsule, composed of polysaccharide and glycoprotein filaments, protects cells from phagocytosis. Either Gram-positive or Gram-negative cells may possess a capsule.
  • The Gram-negative outer membrane regulates nutrient uptake and excludes toxins. The outer membrane contains LPS and protein porins of varying selectivity.
  • The mycobacterial cell wall includes features of both Gram-positive and Gram-negative cells. The arabinogalactan layer adds thickness to the cell wall. The mycolate outer membrane and phenolic glycolipids limit uptake of nutrients and antibiotics.
  • The bacterial cytoskeleton includes proteins that regulate cell size, play a role in determining the rod shape of bacilli, and generate curvature in vibrios.