The prokaryotic cell is simpler than the eukaryotic cell at every level, with one exception: The cell envelope is more complex.
Prokaryotes have no true nuclei; instead they package their DNA in a structure known as the nucleoid. The negatively charged DNA is at least partially neutralized by small polyamines and magnesium ions, but histone-like proteins exist in bacteria and presumably play a role similar to that of histones in eukaryotic chromatin.
Electron micrographs of a typical prokaryotic cell reveal the absence of a nuclear membrane and a mitotic apparatus. The exception to this rule is the planctomycetes, a divergent group of aquatic bacteria, which have a nucleoid surrounded by a nuclear envelope consisting of two membranes. The distinction between prokaryotes and eukaryotes that still holds is that prokaryotes have no eukaryotic-type mitotic apparatus. The nuclear region (Figure 2-6) is filled with DNA fibrils. The nucleoid of most bacterial cells consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million base pairs. However, a few bacteria have been shown to have two, three, or even four dissimilar chromosomes. For example, Vibrio cholerae and Brucella melitensis have two dissimilar chromosomes. There are exceptions to this rule of circularity because some prokaryotes (eg, Borrelia burgdorferi and Streptomyces coelicolor) have been shown to have a linear chromosome.
The nucleoid. A: Color-enhanced transmission electron micrograph of Escherichia coli with the DNA shown in red. (© CNRI/SPL/Photo Researchers, Inc.) B: Chromosome released from a gently lysed cell of E coli. Note how tightly packaged the DNA must be inside the bacterium. (© Dr. Gopal Murti/SPL/Photo Researchers.)
In bacteria, the number of nucleoids, and therefore the number of chromosomes, depend on the growth conditions. Rapidly growing bacteria have more nucleoids per cell than slowly growing ones; however, when multiple copies are present, they are all the same (ie, prokaryotic cells are haploid).
Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplasts; the electron transport enzymes are localized instead in the cytoplasmic membrane. The photosynthetic pigments (carotenoids, bacteriochlorophyll) of photosynthetic bacteria are contained in intracytoplasmic membrane systems of various morphologies. Membrane vesicles (chromatophores) or lamellae are commonly observed membrane types. Some photosynthetic bacteria have specialized nonunit membrane-enclosed structures called chlorosomes. In some Cyanobacteria (formerly known as blue-green algae), the photosynthetic membranes often form multilayered structures known as thylakoids (Figure 2-7). The major accessory pigments used for light harvesting are the phycobilins found on the outer surface of the thylakoid membranes.
Thin section of Synechocystis during division. Many structures are visible. (Reproduced from Stanier RY: The position of cyanobacteria in the world of phototrophs. Carlsberg Res Commun 42:77-98, 1977. With kind permission of Springer + Business Media.)
Bacteria often store reserve materials in the form of insoluble granules, which appear as refractile bodies in the cytoplasm when viewed by phase contrast microscopy. These so-called inclusion bodies almost always function in the storage of energy or as a reservoir of structural building blocks. Most cellular inclusions are bounded by a thin nonunit membrane consisting of lipid, which serves to separate the inclusion from the cytoplasm proper. One of the most common inclusion bodies consists of poly-β-hydroxybutyric acid (PHB), a lipid-like compound consisting of chains of β-hydroxybutyric acid units connected through ester linkages. PHB is produced when the source of nitrogen, sulfur, or phosphorous is limited and there is excess carbon in the medium (Figure 2-8A). Another storage product formed by prokaryotes when carbon is in excess is glycogen, which is a polymer of glucose. PHB and glycogen are used as carbon sources when protein and nucleic acid synthesis are resumed. A variety of prokaryotes are capable of oxidizing reduced sulfur compounds such as hydrogen sulfide and thiosulfate, producing intracellular granules of elemental sulfur (Figure 2-8B). As the reduced sulfur source becomes limiting, the sulfur in the granules is oxidized, usually to sulfate, and the granules slowly disappear. Many bacteria accumulate large reserves of inorganic phosphate in the form of granules of polyphosphate. These granules can be degraded and used as sources of phosphate for nucleic acid and phospholipid synthesis to support growth. These granules are sometimes termed volutin granules or metachromatic granules because they stain red with a blue dye. They are characteristic features of the corynebacteria (see Chapter 13).
Inclusion bodies in bacteria. A: Electron micrograph of Bacillus megaterium (30,500×) showing poly-β-hydroxybutyric acid inclusion body, PHB; cell wall, CW; nucleoid, N; plasma membrane, PM; “mesosome,” M; and ribosomes, R. (Reproduced with permission. © Ralph A. Slepecky/Visuals Unlimited.) B: Cromatium vinosum, a purple sulfur bacterium, with intracellular sulfur granules, bright field microscopy (2000×). (Reproduced with permission from Holt J (editor): The Shorter Bergey’s Manual of Determinative Bacteriology, 8th ed. Williams & Wilkins, 1977. Copyright Bergey’s Manual Trust.)
Certain groups of autotrophic bacteria that fix carbon dioxide to make their biochemical building blocks contain polyhedral bodies surrounded by a protein shell (carboxysomes) containing the key enzyme of CO2 fixation, ribulosebisphosphate carboxylase (see Figure 2-7). Magnetosomes are intracellular crystal particles of the iron mineral magnetite (Fe3O4) that allow certain aquatic bacteria to exhibit magnetotaxis (ie, migration or orientation of the cell with respect to the earth’s magnetic field). Magnetosomes are surrounded by a nonunit membrane containing phospholipids, proteins, and glycoproteins. Gas vesicles are found almost exclusively in microorganisms from aquatic habitats, where they provide buoyancy. The gas vesicle membrane is a 2-nm-thick layer of protein, impermeable to water and solutes but permeable to gases; thus, gas vesicles exist as gas-filled structures surrounded by the constituents of the cytoplasm (Figure 2-9).
Transverse section of a dividing cell of the cyanobacterium Microcystis species showing hexagonal stacking of the cylindric gas vesicles (31,500×). (Micrograph by HS Pankratz. Reproduced with permission from Walsby AE: Gas vesicles. Microbiol Rev 1994;58:94.)
Bacteria contain proteins resembling both the actin and nonactin cytoskeletal proteins of eukaryotic cells as additional proteins that play cytoskeletal roles (Figure 2-10). Actin homologs (eg, MreB, Mbl) perform a variety of functions, helping to determine cell shape, segregate chromosomes, and localize proteins with the cell. Nonactin homologs (eg, FtsZ) and unique bacterial cytoskeletal proteins (eg, SecY, MinD) are involved in determining cell shape and in regulation of cell division and chromosome segregation.
The prokaryotic cytoskeleton. Visualization of the MreB-like cytoskeletal protein (Mbl) of Bacillus subtilis. The Mbl protein has been fused with green fluorescent protein, and live cells have been examined by fluorescence microscopy. A: Arrows point to the helical cytoskeleton cables that extend the length of the cells. B: Three of the cells from A are shown at a higher magnification. (Courtesy of Rut Carballido-Lopez and Jeff Errington.)
Prokaryotic cells are surrounded by complex envelope layers that differ in composition among the major groups. These structures protect the organisms from hostile environments, such as extreme osmolarity, harsh chemicals, and even antibiotics.
The bacterial cell membrane, also called the cytoplasmic membrane, is visible in electron micrographs of thin sections (see Figure 2-15). It is a typical “unit membrane” composed of phospholipids and upward of 200 different kinds of proteins. Proteins account for approximately 70% of the mass of the membrane, which is a considerably higher proportion than that of mammalian cell membranes. Figure 2-11 illustrates a model of membrane organization. The membranes of prokaryotes are distinguished from those of eukaryotic cells by the absence of sterols, the only exception being mycoplasmas that incorporate sterols, such as cholesterol, into their membranes when growing in sterol-containing media.
Bacterial plasma membrane structure. This diagram of the fluid mosaic model of bacterial membrane structure shown the integral proteins (green and red) floating in a lipid bilayer. Peripheral proteins (yellow) are associated loosely with the inner membrane surface. Small spheres represent the hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains. Other membrane lipids such as hopanoids (purple) may be present. For the sake of clarity, phospholipids are shown proportionately much larger size than in real membranes. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ [editors]: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008. © The McGraw-Hill Companies, Inc.)
The cell membranes of the Archaea (see Chapter 1) differ from those of the Bacteria. Some Archaeal cell membranes contain unique lipids, isoprenoids, rather than fatty acids, linked to glycerol by ether rather than an ester linkage. Some of these lipids have no phosphate groups, and therefore, they are not phospholipids. In other species, the cell membrane is made up of a lipid monolayer consisting of long lipids (about twice as long as a phospholipid) with glycerol ethers at both ends (diglycerol tetraethers). The molecules orient themselves with the polar glycerol groups on the surfaces and the nonpolar hydrocarbon chain in the interior. These unusual lipids contribute to the ability of many Archaea to grow under environmental conditions such as high salt, low pH, or very high temperature.
The major functions of the cytoplasmic membrane are (1) selective permeability and transport of solutes; (2) electron transport and oxidative phosphorylation in aerobic species; (3) excretion of hydrolytic exoenzymes; (4) bearing the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids; and (5) bearing the receptors and other proteins of the chemotactic and other sensory transduction systems.
At least 50% of the cytoplasmic membrane must be in the semifluid state for cell growth to occur. At low temperatures, this is achieved by greatly increased synthesis and incorporation of unsaturated fatty acids into the phospholipids of the cell membrane.
1. Permeability and transport—The cytoplasmic membrane forms a hydrophobic barrier impermeable to most hydrophilic molecules. However, several mechanisms (transport systems) exist that enable the cell to transport nutrients into and waste products out of the cell. These transport systems work against a concentration gradient to increase the concentration of nutrients inside the cell, a function that requires energy in some form. There are three general transport mechanisms involved in membrane transport: passive transport, active transport, and group translocation.
a. Passive transport—This mechanism relies on diffusion, uses no energy, and operates only when the solute is at higher concentration outside than inside the cell. Simple diffusion accounts for the entry of very few nutrients, including dissolved oxygen, carbon dioxide, and water itself. Simple diffusion provides neither speed nor selectivity. Facilitated diffusion also uses no energy so the solute never achieves an internal concentration greater than what exists outside the cell. However, facilitated diffusion is selective. Channel proteins form selective channels that facilitate the passage of specific molecules. Facilitated diffusion is common in eukaryotic microorganisms (eg, yeast) but is rare in prokaryotes. Glycerol is one of the few compounds that enters prokaryotic cells by facilitated diffusion.
b. Active transport—Many nutrients are concentrated more than a thousand-fold as a result of active transport. There are two types of active transport mechanisms depending on the source of energy used: ion-coupled transport and ATP-binding cassette (ABC) transport.
1) Ion-coupled transport—These systems move a molecule across the cell membrane at the expense of a previously established ion gradient such as protonmotive or sodium-motive force. There are three basic types: uniport, symport, and antiport (Figure 2-12). Ion-coupled transport is particularly common in aerobic organisms, which have an easier time generating an ion-motive force than do anaerobes. Uniporters catalyze the transport of a substrate independent of any coupled ion. Symporters catalyze the simultaneous transport of two substrates in the same direction by a single carrier; for example, an H+ gradient can permit symport of an oppositely charged ion (eg, glycine) or a neutral molecule (eg, galactose). Antiporters catalyze the simultaneous transport of two like-charged compounds in opposite directions by a common carrier (eg, H+:Na+). Approximately 40% of the substrates transported by E coli use this mechanism.
2) ABC transport—This mechanism uses ATP directly to transport solutes into the cell. In gram-negative bacteria, the transport of many nutrients is facilitated by specific binding proteins located in the periplasmic space; in gram-positive cells, the binding proteins are attached to the outer surface of the cell membrane. These proteins function by transferring the bound substrate to a membrane-bound protein complex. Hydrolysis of ATP is then triggered, and the energy is used to open the membrane pore and allow the unidirectional movement of the substrate into the cell. Approximately 40% of the substrates transported by E coli use this mechanism.
c. Group translocation—In addition to true transport, in which a solute is moved across the membrane without change in structure, bacteria use a process called group translocation (vectorial metabolism) to effect the net uptake of certain sugars (eg, glucose and mannose), the substrate becoming phosphorylated during the transport process. In a strict sense, group translocation is not active transport because no concentration gradient is involved. This process allows bacteria to use their energy resources efficiently by coupling transport with metabolism. In this process, a membrane carrier protein is first phosphorylated in the cytoplasm at the expense of phosphoenolpyruvate; the phosphorylated carrier protein then binds the free sugar at the exterior membrane face and transports it into the cytoplasm, releasing it as sugar phosphate. Such systems of sugar transport are called phosphotransferase systems. Phosphotransferase systems are also involved in movement toward these carbon sources (chemotaxis) and in the regulation of several other metabolic pathways (catabolite repression).
d. Special transport processes—Iron (Fe) is an essential nutrient for the growth of almost all bacteria. Under anaerobic conditions, Fe is generally in the +2 oxidation state and soluble. However, under aerobic conditions, Fe is generally in the +3 oxidation state and insoluble. The internal compartments of animals contain virtually no free Fe; it is sequestered in complexes with such proteins as transferrin and lactoferrin. Some bacteria solve this problem by secreting siderophores—compounds that chelate Fe and promote its transport as a soluble complex. One major group of siderophores consists of derivatives of hydroxamic acid (−CONH2OH), which chelate Fe3+ very strongly. The iron–hydroxamate complex is actively transported into the cell by the cooperative action of a group of proteins that span the outer membrane, periplasm, and inner membrane. The iron is released, and the hydroxamate can exit the cell and be used again for iron transport.
Some pathogenic bacteria use a fundamentally different mechanism involving specific receptors that bind host transferrin and lactoferrin (as well as other iron-containing host proteins). The Fe is removed and transported into the cell by an energy-dependent process.
2. Electron transport and oxidative phosphorylation—The cytochromes and other enzymes and components of the respiratory chain, including certain dehydrogenases, are located in the cell membrane. The bacterial cell membrane is thus a functional analog of the mitochondrial membrane—a relationship which has been taken by many biologists to support the theory that mitochondria have evolved from symbiotic bacteria. The mechanism by which ATP generation is coupled to electron transport is discussed in Chapter 6.
3. Excretion of hydrolytic exoenzymes and pathogenicity proteins—All organisms that rely on macromolecular organic polymers as a source of nutrients (eg, proteins, polysaccharides, lipids) excrete hydrolytic enzymes that degrade the polymers to subunits small enough to penetrate the cell membrane. Higher animals secrete such enzymes into the lumen of the digestive tract; bacteria (both gram positive and gram negative) secrete them directly into the external medium or into the periplasmic space between the peptidoglycan layer and the outer membrane of the cell wall in the case of gram-negative bacteria (see The Cell Wall, later).
In gram-positive bacteria, proteins are secreted directly, but proteins secreted by gram-negative bacteria must traverse the outer membrane as well. Six pathways of protein secretion have been described in bacteria: the type I, type II, type III, type IV, type V, and type VI secretion systems. A schematic overview of the type I to V systems is presented in Figure 2-13. The type I and IV secretion systems have been described in both gram-negative and gram-positive bacteria, but the type II, III, V, and VI secretion systems have been found only in gram-negative bacteria. Proteins secreted by the type I and III pathways traverse the inner membrane (IM) and outer membrane (OM) in one step, but proteins secreted by the type II and V pathways cross the IM and OM in separate steps. Proteins secreted by the type II and V pathways are synthesized on cytoplasmic ribosomes as preproteins containing an extra leader or signal sequence of 15–40 amino acids—most commonly about 30 amino acids—at the amino terminal and require the sec system for transport across the IM. In E coli, the sec pathway comprises a number of IM proteins (SecD to SecF, SecY), a cell membrane–associated ATPase (SecA) that provides energy for export, a chaperone (SecB) that binds to the preprotein, and the periplasmic signal peptidase. After translocation, the leader sequence is cleaved off by the membrane-bound signal peptidase, and the mature protein is released into the periplasmic space. In contrast, proteins secreted by the type I and III systems do not have a leader sequence and are exported intact.
In gram-negative and gram-positive bacteria, another plasma membrane translocation system, called the tat pathway, can move proteins across the plasma membrane. In gram-negative bacteria, these proteins are then delivered to the type II system (Figure 2-13). The tat pathway is distinct from the sec system in that it translocates already folded proteins.
Although proteins secreted by the type II and V systems are similar in the mechanism by which they cross the IM, differences exist in how they traverse the OM. Proteins secreted by the type II system are transported across the OM by a multiprotein complex (see Figure 2-13). This is the primary pathway for the secretion of extracellular degradative enzymes by gram-negative bacteria. Elastase, phospholipase C, and exotoxin A are secreted by this system in Pseudomonas aeruginosa. However, proteins secreted by the type V system autotransport across the outer membrane by virtue of a carboxyl terminal sequence, which is enzymatically removed upon release of the protein from the OM. Some extracellular proteins—eg, the IgA protease of Neisseria gonorrhoeae and the vacuolating cytotoxin of Helicobacter pylori—are secreted by this system.
The type I and III secretion pathways are sec independent and thus do not involve amino terminal processing of the secreted proteins. Protein secretion by these pathways occurs in a continuous process without the presence of a cytoplasmic intermediate. Type I secretion is exemplified by the α-hemolysin of E coli and the adenylyl cyclase of Bordetella pertussis. Type I secretion requires three secretory proteins: an IM ATP-binding cassette (ABC transporter), which provides energy for protein secretion; an OM protein; and a membrane fusion protein, which is anchored in the inner membrane and spans the periplasmic space (see Figure 2-13). Instead of a signal peptide, the information is located within the carboxyl terminal 60 amino acids of the secreted protein.
The type III secretion pathway is a contact-dependent system. It is activated by contact with a host cell, and then injects a toxin protein into the host cell directly. The type III secretion apparatus is composed of approximately 20 proteins, most of which are located in the IM. Most of these IM components are homologous to the flagellar biosynthesis apparatus of both gram-negative and gram-positive bacteria. As in type I secretion, the proteins secreted via the type III pathway are not subject to amino terminal processing during secretion.
Type IV pathways secrete either polypeptide toxins (directed against eukaryotic cells) or protein–DNA complexes either between two bacterial cells or between a bacterial and a eukaryotic cell. Type IV secretion is exemplified by the protein–DNA complex delivered by Agrobacterium tumefaciens into a plant cell. Additionally, B pertussis and H pylori possess type IV secretion systems that mediate secretion of pertussis toxin and interleukin-8–inducing factor, respectively. The sec-independent type VI secretion was recently described in P aeruginosa, where it contributes to pathogenicity in patients with cystic fibrosis. This secretion system is composed of 15–20 proteins whose biochemical functions are not well understood. However, recent studies suggest that some of these proteins share homology with bacteriophage tail proteins.
The characteristics of the protein secretion systems of bacteria are summarized in Table 9-5.
4. Biosynthetic functions—The cell membrane is the site of the carrier lipids on which the subunits of the cell wall are assembled (see the discussion of synthesis of cell wall substances in Chapter 6) as well as of the enzymes of cell wall biosynthesis. The enzymes of phospholipid synthesis are also localized in the cell membrane.
5. Chemotactic systems—Attractants and repellents bind to specific receptors in the bacterial membrane (see Flagella, later). There are at least 20 different chemoreceptors in the membrane of E coli, some of which also function as a first step in the transport process.
Three types of porters: A: uniporters, B: symporters, and C: antiporters. Uniporters catalyze the transport of a single species independently of any other, symporters catalyze the cotransport of two dissimilar species (usually a solute and a positively charged ion, H+) in the same direction, and antiporters catalyze the exchange transport of two similar solutes in opposite directions. A single transport protein may catalyze just one of these processes, two of these processes, or even all three of these processes, depending on conditions. Uniporters, symporters, and antiporters have been found to be structurally similar and evolutionarily related, and they function by similar mechanisms. (Reproduced with permission from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.)
The protein secretion systems of gram-negative bacteria. Five secretion systems of gram-negative bacteria are shown. The Sec-dependent and Tat pathways deliver proteins from the cytoplasm to the periplasmic space. The type II, type V, and sometimes type IV systems complete the secretion process begun by the Sec-dependent pathway. The Tat system appears to deliver proteins only to the type II pathway. The type I and III systems bypass the Sec-dependent and Tat pathways, moving proteins directly from the cytoplasm, through the outer membrane, to the extracellular space. The type IV system can work either with the Sec-dependent pathway or can work alone to transport proteins to the extracellular space. Proteins translocated by the Sec-dependent pathway and the type III pathway are delivered to those systems by chaperone proteins. ADP, adenosine diphosphate; ATP, adenosine triphosphate; EFGY; PuIS; SecD; TolC; Yop. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ [editors]: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008. © The McGraw-Hill Companies, Inc.)
The internal osmotic pressure of most bacteria ranges from 5 to 20 atm as a result of solute concentration via active transport. In most environments, this pressure would be sufficient to burst the cell were it not for the presence of a high-tensile-strength cell wall (Figure 2-14). The bacterial cell wall owes its strength to a layer composed of a substance variously referred to as murein, mucopeptide, or peptidoglycan (all are synonyms). The structure of peptidoglycan is discussed as follows.
The rigid cell wall determines the shape of the bacterium. Even though the cell has split apart, the cell wall maintains it’s original shape. (Courtesy of Dale C. Birdsell.)
Most bacteria are classified as gram positive or gram negative according to their response to the Gram-staining procedure. This procedure was named for the histologist Hans Christian Gram, who developed this differential staining procedure in an attempt to stain bacteria in infected tissues. The Gram stain depends on the ability of certain bacteria (the gram-positive bacteria) to retain a complex of crystal violet (a purple dye) and iodine after a brief wash with alcohol or acetone. Gram-negative bacteria do not retain the dye–iodine complex and become translucent, but they can then be counterstained with safranin (a red dye). Thus, gram-positive bacteria look purple under the microscope, and gram-negative bacteria look red. The distinction between these two groups turns out to reflect fundamental differences in their cell envelopes (Table 2-1).
In addition to giving osmotic protection, the cell wall plays an essential role in cell division as well as serving as a primer for its own biosynthesis. Various layers of the wall are the sites of major antigenic determinants of the cell surface, and one component—the lipopolysaccharide of gram-negative cell walls—is responsible for the nonspecific endotoxin activity of gram-negative bacteria. The cell wall is, in general, nonselectively permeable; one layer of the gram-negative wall, however—the outer membrane—hinders the passage of relatively large molecules (see below).
The biosynthesis of the cell wall and the antibiotics that interfere with this process are discussed in Chapter 6.
A. The Peptidoglycan Layer
Peptidoglycan is a complex polymer consisting, for the purposes of description, of three parts: a backbone, composed of alternating N-acetylglucosamine and N-acetylmuramic acid connected by β1→4 linkages; a set of identical tetrapeptide side chains attached to N-acetylmuramic acid; and a set of identical peptide cross-bridges (Figure 2-15). The backbone is the same in all bacterial species; the tetrapeptide side chains and the peptide cross-bridges vary from species to species. In many gram-negative cell walls, the cross-bridge consists of a direct peptide linkage between the diaminopimelic acid (DAP) amino group of one side chain and the carboxyl group of the terminal d-alanine of a second side chain.
Components and structure of peptidoglycan. A: Chemical structure of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM); the ring structures of the two molecules are glucose. Glycan chains are composed of alternating subunits of NAG and NAM joined by covalent bonds. Adjacent glycan chains are cross-linked via their tetrapeptide chains to create peptidoglycan. B: Interconnected glycan chains form a very large three-dimensional molecule of peptidoglycan. The β1→4 linkages in the backbone are cleaved by lysozyme. (Reproduced with permission from Nester EW, Anderson DG, Roberts CE, Nester MT: Microbiology: A Human Perspective, 6th ed. McGraw-Hill; 2009.)
The tetrapeptide side chains of all species, however, have certain important features in common. Most have l-alanine at position 1 (attached to N-acetylmuramic acid), d-glutamate or substituted d-glutamate at position 2, and d-alanine at position 4. Position 3 is the most variable one: Most gram-negative bacteria have diaminopimelic acid at this position, to which is linked the lipoprotein cell wall component discussed as follows. Gram-positive bacteria usually have l-lysine at position 3; however, some may have diaminopimelic acid or another amino acid at this position.
Diaminopimelic acid is a unique element of bacterial cell walls. It is never found in the cell walls of Archaea or eukaryotes. Diaminopimelic acid is the immediate precursor of lysine in the bacterial biosynthesis of that amino acid (see Figure 6-19). Bacterial mutants that are blocked before diaminopimelic acid in the biosynthetic pathway grow normally when provided with diaminopimelic acid in the medium; when given l-lysine alone, however, they lyse, because they continue to grow but are specifically unable to make new cell wall peptidoglycan.
The fact that all peptidoglycan chains are cross-linked means that each peptidoglycan layer is a single giant molecule. In gram-positive bacteria, there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall material; in gram-negative bacteria, there appears to be only one or two sheets, comprising 5–10% of the wall material. Bacteria owe their shapes, which are characteristic of particular species, to their cell wall structure.
B. Special Components of Gram-Positive Cell Walls
Most gram-positive cell walls contain considerable amounts of teichoic and teichuronic acids, which may account for up to 50% of the dry weight of the wall and 10% of the dry weight of the total cell. In addition, some gram-positive walls may contain polysaccharide molecules.
1. Teichoic and teichuronic acids—The term teichoic acids encompasses all wall, membrane, or capsular polymers containing glycerophosphate or ribitol phosphate residues. These polyalcohols are connected by phosphodiester linkages and usually have other sugars and d-alanine attached (Figure 2-16A). Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. There are two types of teichoic acids: wall teichoic acid (WTA), covalently linked to peptidoglycan; and membrane teichoic acid, covalently linked to membrane glycolipid. Because the latter are intimately associated with lipids, they have been called lipoteichoic acids (LTA). Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. Although not all gram-positive bacteria have conventional LTA and WTA, those that lack these polymers generally have functionally similar ones.
Most teichoic acids contain large amounts of d-alanine, usually attached to position 2 or 3 of glycerol or position 3 or 4 of ribitol. In some of the more complex teichoic acids, however, d-alanine is attached to one of the sugar residues. In addition to d-alanine, other substituents may be attached to the free hydroxyl groups of glycerol and ribitol (eg, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, or succinate). A given species may have more than one type of sugar substituent in addition to d-alanine; in such cases, it is not certain whether the different sugars occur on the same or on separate teichoic acid molecules. The composition of the teichoic acid formed by a given bacterial species can vary with the composition of the growth medium.
The teichoic acids constitute major surface antigens of those gram-positive species that possess them, and their accessibility to antibodies has been taken as evidence that they lie on the outside surface of the peptidoglycan. Their activity is often increased, however, by partial digestion of the peptidoglycan; thus, much of the teichoic acid may lie between the cytoplasmic membrane and the peptidoglycan layer, possibly extending upward through pores in the latter (Figure 2-16B). In the pneumococcus (Streptococcus pneumoniae), the teichoic acids bear the antigenic determinants called Forssman antigen. In Streptococcus pyogenes, LTA is associated with the M protein that protrudes from the cell membrane through the peptidoglycan layer. The long M protein molecules together with the LTA form microfibrils that facilitate the attachment of S pyogenes to animal cells (see Chapter 14).
The teichuronic acids are similar polymers, but the repeat units include sugar acids (eg, N-acetylmannosuronic or d-glucosuronic acid) instead of phosphoric acids. They are synthesized in place of teichoic acids when phosphate is limiting.
2. Polysaccharides—The hydrolysis of gram-positive walls has yielded, from certain species, neutral sugars such as mannose, arabinose, rhamnose, and glucosamine and acidic sugars such as glucuronic acid and mannuronic acid. It has been proposed that these sugars exist as subunits of polysaccharides in the cell wall; the discovery, however, that teichoic and teichuronic acids may contain a variety of sugars (see Figure 2-16A) leaves the true origin of these sugars uncertain.
A: Teichoic acid structure. The segment of a teichoic acid made of phosphate, glycerol, and a side chain, R. R may represent d-alanine, glucose, or other molecules. B: Teichoic and lipoteichoic acids of the gram-positive envelope. (Reproduced with permission from Willey JM, Sherwood LM, Woolverton CJ [editors]: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008.)
C. Special Components of Gram-Negative Cell Walls
Gram-negative cell walls contain three components that lie outside of the peptidoglycan layer: lipoprotein, outer membrane, and lipopolysaccharide (Figure 2-17).
Molecular representation of the envelope of a gram-negative bacterium. Ovals and rectangles represent sugar residues, and circles depict the polar head groups of the glycerophospholipids (phosphatidylethanolamine and phosphatidylglycerol). The core region shown is that of Escherichia coli K-12, a strain that does not normally contain an O-antigen repeat unless transformed with an appropriate plasmid. MDO, membrane-derived oligosaccharides. (Reproduced with permission from Raetz CRH: Bacterial endotoxins: Extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol 1993;175:5745.)
1. Outer membrane—The outer membrane is chemically distinct from all other biological membranes. It is a bilayered structure; its inner leaflet resembles in composition that of the cell membrane, and its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS) (see below). As a result, the leaflets of this membrane are asymmetrical, and the properties of this bilayer differ considerably from those of a symmetrical biologic membrane such as the cell membrane.
The ability of the outer membrane to exclude hydrophobic molecules is an unusual feature among biologic membranes and serves to protect the cell (in the case of enteric bacteria) from deleterious substances such as bile salts. Because of its lipid nature, the outer membrane would be expected to exclude hydrophilic molecules as well. However, the outer membrane has special channels, consisting of protein molecules called porins that permit the passive diffusion of low-molecular-weight hydrophilic compounds such as sugars, amino acids, and certain ions. Large antibiotic molecules penetrate the outer membrane relatively slowly, which accounts for the relatively high antibiotic resistance of gram-negative bacteria. The permeability of the outer membrane varies widely from one gram-negative species to another; in P aeruginosa, for example, which is extremely resistant to antibacterial agents, the outer membrane is 100 times less permeable than that of E coli.
The major proteins of the outer membrane, named according to the genes that code for them, have been placed into several functional categories on the basis of mutants in which they are lacking and on the basis of experiments in which purified proteins have been reconstituted into artificial membranes. Porins, exemplified by OmpC, D, and F and PhoE of E coli and Salmonella typhimurium, are trimeric proteins that penetrate both faces of the outer membrane (Figure 2-18). They form relatively nonspecific pores that permit the free diffusion of small hydrophilic solutes across the membrane. The porins of different species have different exclusion limits, ranging from molecular weights of about 600 in E coli and S typhimurium to more than 3000 in P aeruginosa.
Members of a second group of outer membrane proteins, which resemble porins in many ways, are exemplified by LamB and Tsx. LamB, an inducible porin that is also the receptor for lambda bacteriophage, is responsible for most of the transmembrane diffusion of maltose and maltodextrins; Tsx, the receptor for T6 bacteriophage, is responsible for the transmembrane diffusion of nucleosides and some amino acids. LamB allows some passage of other solutes; however, its relative specificity may reflect weak interactions of solutes with configuration-specific sites within the channel.
The OmpA protein is an abundant protein in the outer membrane. The OmpA protein participates in the anchoring of the outer membrane to the peptidoglycan layer; it is also the sex pilus receptor in F-mediated bacterial conjugation (see Chapter 7).
The outer membrane also contains a set of less abundant proteins that are involved in the transport of specific molecules such as vitamin B12 and iron-siderophore complexes. They show high affinity for their substrates and probably function like the classic carrier transport systems of the cytoplasmic membrane. The proper function of these proteins requires energy coupled through a protein called TonB. Additional minor proteins include a limited number of enzymes, among them phospholipases and proteases.
The topology of the major proteins of the outer membrane, based on cross-linking studies and analyses of functional relationships, is shown in Figure 2-17. The outer membrane is connected to both the peptidoglycan layer and the cytoplasmic membrane. The connection with the peptidoglycan layer is primarily mediated by the outer membrane lipoprotein (see below). About one-third of the lipoprotein molecules are covalently linked to peptidoglycan and help hold the two structures together. A noncovalent association of some of the porins with the peptidoglycan layer plays a lesser role in connecting the outer membrane with this structure. Outer membrane proteins are synthesized on ribosomes bound to the cytoplasmic surface of the cell membrane; how they are transferred to the outer membrane is still uncertain, but one hypothesis suggests that transfer occurs at zones of adhesion between the cytoplasmic and outer membranes, which are visible in the electron microscope. Unfortunately, firm evidence for such areas of adhesion has proven hard to come by.
2. Lipopolysaccharide (LPS)—The LPS of gram-negative cell walls consists of a complex glycolipid, called lipid A, to which is attached a polysaccharide made up of a core and a terminal series of repeat units (Figure 2-19A). The lipid A component is embedded in the outer leaflet of the membrane anchoring the LPS. LPS is synthesized on the cytoplasmic membrane and transported to its final exterior position. The presence of LPS is required for the function of many outer membrane proteins.
Lipid A consists of phosphorylated glucosamine disaccharide units to which are attached a number of long-chain fatty acids (Figure 2-19). β-Hydroxymyristic acid, a C14 fatty acid, is always present and is unique to this lipid; the other fatty acids, along with substituent groups on the phosphates, vary according to the bacterial species.
The polysaccharide core, shown in Figure 2-19A and B, is similar in all gram-negative species that have LPS and includes two characteristic sugars, ketodeoxyoctanoic acid (KDO) and a heptose. Each species, however, contains a unique repeat unit, that of Salmonella being shown in Figure 2-19A. The repeat units are usually linear trisaccharides or branched tetra- or pentasaccharides. The repeat unit is referred to as the O antigen. The hydrophilic carbohydrate chains of the O antigen cover the bacterial surface and exclude hydrophobic compounds.
The negatively charged LPS molecules are noncovalently cross-bridged by divalent cations (ie, Ca2+ and Mg2+); this stabilizes the membrane and provides a barrier to hydrophobic molecules. Removal of the divalent cations with chelating agents or their displacement by polycationic antibiotics such as polymyxins and aminoglycosides renders the outer membrane permeable to large hydrophobic molecules.
Lipopolysaccharide, which is extremely toxic to animals, has been called the endotoxin of gram-negative bacteria because it is firmly bound to the cell surface and is released only when the cells are lysed. When LPS is split into lipid A and polysaccharide, all of the toxicity is associated with the former. The O antigen is highly immunogenic in a vertebrate animal. Antigenic specificity is conferred by the O antigen because this antigen is highly variable among species and even in strains within a species. The number of possible antigenic types is very great: Over 1000 have been recognized in Salmonella alone. Not all gram-negative bacteria have outer membrane LPS composed of a variable number of repeated oligosaccharide units (see Figure 2-19); the outer membrane glycolipids of bacteria that colonize mucosal surfaces (eg, Neisseria meningitidis, N gonorrhoeae, Haemophilus influenzae, and Haemophilus ducreyi) have relatively short, multiantennary (ie, branched) glycans. These smaller glycolipids have been compared with the “R-type” truncated LPS structures, which lack O antigens and are produced by rough mutants of enteric bacteria such as E coli. However, their structures more closely resemble those of the glycosphingolipids of mammalian cell membranes, and they are more properly termed lipooligosaccharides (LOS). These molecules exhibit extensive antigenic and structural diversity even within a single strain. LOS is an important virulence factor. Epitopes have been identified on LOS that mimic host structures and may enable these organisms to evade the immune response of the host. Some LOS (eg, those from N gonorrhoeae, N meningitidis, and H ducreyi) have a terminal N-acetyllactosamine (Galβ-1→4-GlcNAc) residue that is immunochemically similar to the precursor of the human erythrocyte i antigen. In the presence of a bacterial enzyme called sialyltransferase and a host or bacterial substrate (cytidine monophospho-N-acetylneuraminic acid, CMP-NANA), the N-acetyllactosamine residue is sialylated. This sialylation, which occurs in vivo, provides the organism with the environmental advantages of molecular mimicry of a host antigen and the biologic masking thought to be provided by sialic acids.
3. Lipoprotein—Molecules of an unusual lipoprotein cross-link the outer membrane and peptidoglycan layers (see Figure 2-17). The lipoprotein contains 57 amino acids, representing repeats of a 15-amino-acid sequence; it is peptide-linked to DAP residues of the peptidoglycan tetrapeptide side chains. The lipid component, consisting of a diglyceride thioether linked to a terminal cysteine, is noncovalently inserted in the outer membrane. Lipoprotein is numerically the most abundant protein of gram-negative cells (ca 700,000 molecules per cell). Its function (inferred from the behavior of mutants that lack it) is to stabilize the outer membrane and anchor it to the peptidoglycan layer.
4. The periplasmic space—The space between the inner and outer membranes, called the periplasmic space, contains the peptidoglycan layer and a gel-like solution of proteins. The periplasmic space is approximately 20–40% of the cell volume, which is far from insignificant. The periplasmic proteins include binding proteins for specific substrates (eg, amino acids, sugars, vitamins, and ions), hydrolytic enzymes (eg, alkaline phosphatase and 5′-nucleotidase) that break down nontransportable substrates into transportable ones, and detoxifying enzymes (eg, β-lactamase and aminoglycoside-phosphorylase) that inactivate certain antibiotics. The periplasm also contains high concentrations of highly branched polymers of d-glucose, 8 to 10 residues long, which are variously substituted with glycerol phosphate and phosphatidylethanolamine residues; some contain O-succinyl esters. These so-called membrane-derived oligosaccharides appear to play a role in osmoregulation because cells grown in media of low osmolarity increase their synthesis of these compounds 16-fold.
A: General fold of a porin monomer (OmpF porin from Escherichia coli). The large hollow β-barrel structure is formed by antiparallel arrangement of 16 β-strands. The strands are connected by short loops or regular turns on the periplasmic rim (bottom), and long irregular loops face the cell exterior (top). The internal loop, which connects β-strands 5 and 6 and extends inside the barrel, is highlighted in dark. The chain terminals are marked. The surface closest to the viewer is involved in subunit contacts. B: Schematic representation of the OmpF trimer. The view is from the extracellular space along the molecular threefold symmetry axis. (Reproduced with permission from Schirmer T: General and specific porins from bacterial outer membranes. J Struct Biol 1998;121:101.)
Lipopolysaccharide structure. A: The lipopolysaccharide from Salmonella. This slightly simplified diagram illustrates one form of the LPS. Abe, abequose; Gal, galactose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine; P, phosphate; Rha, l-rhamnose. Lipid A is buried in the outer membrane. B: Molecular model of an Escherichia coli lipopolysaccharide. The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in this model. (Reproduced with permission from Willey VM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill, 2008. © The McGraw-Hill Companies, Inc.)
D. The Acid-Fast Cell Wall
Some bacteria, notably the tubercle bacillus (M tuberculosis) and its relatives have cell walls that contain large amounts of waxes, complex branched hydrocarbons (70–90 carbons long) known as mycolic acids. The cell wall is composed of peptidoglycan and an external asymmetric lipid bilayer; the inner leaflet contains mycolic acids linked to an arabinoglycan, and the outer leaflet contains other extractable lipids. This is a highly ordered lipid bilayer in which proteins are embedded, forming water-filled pores through which nutrients and certain drugs can pass slowly. Some compounds can also penetrate the lipid domains of the cell wall albeit slowly. This hydrophobic structure renders these bacteria resistant to many harsh chemicals, including detergents and strong acids. If a dye is introduced into these cells by brief heating or treatment with detergents, it cannot be removed by dilute hydrochloric acid, as in other bacteria. These organisms are therefore called acid fast. The permeability of the cell wall to hydrophilic molecules is 100- to 1000-fold lower than for E coli and may be responsible for the slow growth rate of mycobacteria.
E. Cell Walls of the Archaea
The Archaea do not have cell walls like the Bacteria. Some have a simple S-layer (see below) often composed of glycoproteins. Some Archaea have a rigid cell wall composed of polysaccharides or a peptidoglycan called pseudomurein. The pseudomurein differs from the peptidoglycan of bacteria by having l-amino acids rather than d-amino acids and disaccharide units with an α-1→3 rather than a β1→4 linkage. Archaea that have a pseudomurein cell wall are gram positive.
F. Crystalline Surface Layers
Many bacteria, both gram-positive and gram-negative bacteria as well as Archaebacteria, possess a two-dimensional crystalline, subunit-type layer lattice of protein or glycoprotein molecules (S-layer) as the outermost component of the cell envelope. In both gram-positive and gram-negative bacteria, this structure is sometimes several molecules thick. In some Archaea, they are the only layer external to the cell membrane.
S-layers are generally composed of a single kind of protein molecule, sometimes with carbohydrates attached. The isolated molecules are capable of self-assembly (ie, they make sheets similar or identical to those present on the cells). S-layer proteins are resistant to proteolytic enzymes and protein-denaturing agents. The function of the S-layer is uncertain but is probably protective. In some cases, it has been shown to protect the cell from wall-degrading enzymes, from invasion by Bdellovibrio bacteriovorous (a predatory bacterium), and from bacteriophages. It also plays a role in the maintenance of cell shape in some species of Archaebacteria, and it may be involved in cell adhesion to host epidermal surfaces.
G. Enzymes That Attack Cell Walls
The β1→4 linkage of the peptidoglycan backbone is hydrolyzed by the enzyme lysozyme (see Figure 2-15), which is found in animal secretions (tears, saliva, nasal secretions) as well as in egg white. Gram-positive bacteria treated with lysozyme in low-osmotic-strength media lyse; if the osmotic strength of the medium is raised to balance the internal osmotic pressure of the cell, free spherical bodies called protoplasts are liberated. The outer membrane of the gram-negative cell wall prevents access of lysozyme unless disrupted by an agent such as ethylene-diaminetetraacetic acid (EDTA), a compound that chelates divalent cations; in osmotically protected media, cells treated with EDTA-lysozyme form spheroplasts that still possess remnants of the complex gram-negative wall, including the outer membrane.
Bacteria themselves possess a number of autolysins, hydrolytic enzymes that attack peptidoglycan, including muramidases, glucosaminidases, endopeptidases, and carboxypeptidases. These enzymes catalyze the turnover or degradation of peptidoglycan in bacteria. These enzymes presumably participate in cell wall growth and turnover and in cell separation, but their activity is most apparent during the dissolution of dead cells (autolysis).
Enzymes that degrade bacterial cell walls are also found in cells that digest whole bacteria (eg, protozoa and the phagocytic cells of higher animals).
Cell wall synthesis is necessary for cell division; however, the incorporation of new cell wall material varies with the shape of the bacterium. Rod-shaped bacteria (eg, E coli, Bacillus subtilis) have two modes of cell wall synthesis; new peptidoglycan is inserted along a helical path leading to elongation of the cell and is inserted in a closing ring around the future division site, leading to the formation of the division septum. Coccoid cells such as S aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division site. A third form of cell wall growth is exemplified by S pneumoniae, which are not true cocci, because their shape is not totally round but instead have the shape of a rugby ball. S pneumoniae synthesizes cell wall not only at the septum but also at the so-called equatorial rings (Figure 2-20).
Incorporation of new cell wall in differently shaped bacteria. Rod-shaped bacteria such as Bacillus subtilis or Escherichia coli have two modes of cell wall synthesis: New peptidoglycan is inserted along a helical path (A), leading to elongation of the lateral wall and is inserted in a closing ring around the future division site, leading to the formation of the division septum (B). Streptococcus pneumoniae cells have the shape of a rugby ball and elongate by inserting new cell wall material at the so-called equatorial rings (A), which correspond to an outgrowth of the cell wall that encircles the cell. An initial ring is duplicated, and the two resultant rings are progressively separated, marking the future division sites of the daughter cells. The division septum is then synthesized in the middle of the cell (B). Round cells such as Staphylococcus aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division septum (B). (Reproduced with permission from Scheffers DJ, Pinho MG: Bacterial cell wall synthesis: new insights from localization studies. Microbiol Mol Biol Rev 2005;69:585.)
I. Protoplasts, Spheroplasts, and L Forms
Removal of the bacterial wall may be accomplished by hydrolysis with lysozyme or by blocking peptidoglycan synthesis with an antibiotic such as penicillin. In osmotically protective media, such treatments liberate protoplasts from gram-positive cells and spheroplasts (which retain outer membrane and entrapped peptidoglycan) from gram-negative cells.
If such cells are able to grow and divide, they are called L forms. L forms are difficult to cultivate and usually require a medium that is solidified with agar as well as having the right osmotic strength. L forms are produced more readily with penicillin than with lysozyme, suggesting the need for residual peptidoglycan.
Some L forms can revert to the normal bacillary form upon removal of the inducing stimulus. Thus, they are able to resume normal cell wall synthesis. Others are stable and never revert. The factor that determines their capacity to revert may again be the presence of residual peptidoglycan, which normally acts as a primer in its own biosynthesis.
Some bacterial species produce L forms spontaneously. The spontaneous or antibiotic-induced formation of L forms in the host may produce chronic infections, the organisms persisting by becoming sequestered in protective regions of the body. Because L-form infections are relatively resistant to antibiotic treatment, they present special problems in chemotherapy. Their reversion to the bacillary form can produce relapses of the overt infection.
The mycoplasmas are cell wall–lacking bacteria containing no peptidoglycan (see Figure 25-1). There are also wall-less Archaea, but they have been less well studied. Genomic analysis places the mycoplasmas close to the gram-positive bacteria from which they may have been derived. Mycoplasmas lack a target for cell wall–inhibiting antimicrobial agents (eg, penicillins and cephalosporins) and are therefore resistant to these drugs. Some, such as Mycoplasma pneumoniae, an agent of pneumonia, contain sterols in their membranes. The difference between L forms and mycoplasmas is that when the murein is allowed to reform, L forms revert to their original bacteria shape, but mycoplasmas never do.
Many bacteria synthesize large amounts of extracellular polymer when growing in their natural environments. With one known exception (the poly-d-glutamic acid capsules of Bacillus anthracis and Bacillus licheniformis), the extracellular material is polysaccharide (Table 2-2). The terms capsule and slime layer are frequently used to describe polysaccharide layers; the more inclusive term glycocalyx is also used. Glycocalyx is defined as the polysaccharide-containing material lying outside the cell. A condensed, well-defined layer closely surrounding the cell that excludes particles, such as India ink, is referred to as a capsule (Figure 2-21). If the glycocalyx is loosely associated with the cell and does not exclude particles, it is referred to as a slime layer. Extracellular polymer is synthesized by enzymes located at the surface of the bacterial cell. Streptococcus mutans, for example, uses two enzymes—glucosyl transferase and fructosyl transferase—to synthesize long-chain dextrans (poly-d-glucose) and levans (poly-d-fructose) from sucrose. These polymers are called homopolymers. Polymers containing more than one kind of monosaccharide are called heteropolymers.
TABLE 2-2Chemical Composition of the Extracellular Polymer in Selected Bacteria |Favorite Table|Download (.pdf) TABLE 2-2 Chemical Composition of the Extracellular Polymer in Selected Bacteria
|Organism ||Polymer ||Chemical Subunits |
|Bacillus anthracis ||Polypeptide ||d-Glutamic acid |
|Enterobacter aerogenes ||Complex polysaccharide ||Glucose, fucose, glucuronic acid |
|Haemophilus influenzae ||Serogroup b ||Ribose, ribitol, phosphate |
|Neisseria meningitidis ||Homopolymers and heteropolymers, eg, || |
| ||Serogroup A ||Partially O-acetylated N-acetylmannosaminephosphate |
| ||Serogroup B ||N-Acetylneuraminic acid (sialic acid) |
| ||Serogroup C ||Acetylated sialic acid |
| ||Serogroup 135 ||Galactose, sialic acid |
|Pseudomonas aeruginosa ||Alginate ||d-Manuronic acid, l-glucuronic acid |
|Streptococcus pneumoniae ||Complex polysaccharide (many types), eg, || |
|(pneumococcus) ||Type II ||Rhamnose, glucose, glucuronic acid |
| ||Type III ||Glucose, glucuronic acid |
| ||Type VI ||Galactose, glucose, rhamnose |
| ||Type XIV ||Galactose, glucose, N-acetylglucosamine |
| ||Type XVIII ||Rhamnose, glucose |
|Streptococcus pyogenes (group A) ||Hyaluronic acid ||N-Acetylglucosamine, glucuronic acid |
|Streptococcus salivarius ||Levan ||Fructose |
Bacterial capsules. A: Bacillus anthracis M’Faydean capsule stain, grown at 35°C, in defibrinated horse blood. B: Demonstration of the presence of a capsule in B anthracis by negative staining with India ink. This method is useful for improving visualization of encapsulated bacteria in clinical samples such as blood, blood culture bottles, or cerebrospinal fluid. (CDC, courtesy of Larry Stauffer, Oregon State Public Health Laboratory.)
The capsule contributes to the invasiveness of pathogenic bacteria—encapsulated cells are protected from phagocytosis unless they are coated with anticapsular antibody. The glycocalyx plays a role in the adherence of bacteria to surfaces in their environment, including the cells of plant and animal hosts. S mutans, for example, owes its capacity to adhere tightly to tooth enamel to its glycocalyx. Bacterial cells of the same or different species become entrapped in the glycocalyx, which forms the layer known as plaque on the tooth surface; acidic products excreted by these bacteria cause dental caries (see Chapter 10). The essential role of the glycocalyx in this process—and its formation from sucrose—explains the correlation of dental caries with sucrose consumption by the human population. Because outer polysaccharide layers bind a significant amount of water, the glycocalyx layer may also play a role in resistance to desiccation.
Bacterial flagella are thread-like appendages composed entirely of protein, 12–30 nm in diameter. They are the organs of locomotion for the forms that possess them. Three types of arrangement are known: monotrichous (single polar flagellum), lophotrichous (multiple polar flagella), and peritrichous (flagella distributed over the entire cell). The three types are illustrated in Figure 2-22.
Bacterial flagellation. A: Vibrio metchnikovii, a monotrichous bacterium (7500×). (Reproduced with permission from van Iterson W: Biochim Biophys Acta 1947;1:527.) B: Electron micrograph of Spirillum serpens, showing lophotrichous flagellation (9000×). (Reproduced with permission from van Iterson W: Biochim Biophys Acta 1947;1:527.) C: Electron micrograph of Proteus vulgaris, showing peritrichous flagellation (9000×). Note basal granules. (Reproduced with permission from Houwink A, van Iterson W: Electron microscopical observations on bacterial cytology; a study on flagellation. Biochim Biophys Acta 1950;5:10.)
A bacterial flagellum is made up of several thousand molecules of a protein subunit called flagellin. In a few organisms (eg, Caulobacter species), flagella are composed of two types of flagellin, but in most, only a single type is found. The flagellum is formed by the aggregation of subunits to form a helical structure. If flagella are removed by mechanically agitating a suspension of bacteria, new flagella are rapidly formed by the synthesis, aggregation, and extrusion of flagellin subunits; motility is restored within 3–6 minutes. The flagellins of different bacterial species presumably differ from one another in primary structure. They are highly antigenic (H antigens), and some of the immune responses to infection are directed against these proteins.
The flagellum is attached to the bacterial cell body by a complex structure consisting of a hook and a basal body. The hook is a short curved structure that appears to act as the universal joint between the motor in the basal structure and the flagellum. The basal body bears a set of rings, one pair in gram-positive bacteria and two pairs in gram-negative bacteria. An interpretative diagram of the gram-negative structure is shown in Figure 2-23; the rings labeled L and P are absent in gram-positive cells. The complexity of the bacterial flagellum is revealed by genetic studies, which show that over 40 gene products are involved in its assembly and function.
A: General structure of the flagellum of a gram-negative bacterium, such as Escherichia coli or Salmonella typhimurium. The filament-hook-basal body complex has been isolated and extensively characterized. The location of the export apparatus has not been demonstrated. B: An exploded diagram of the flagellum showing the substructures and the proteins from which they are constructed. The FliF protein is responsible for the M-ring feature, S-ring feature, and collar feature of the substructure shown, which is collectively termed the MS ring. The location of FliE with respect to the MS ring and the rod—and the order of the FlgB, FlgC, and FlgF proteins within the proximal rod—is not known. (From Macnab RM: Genetics and biogenesis of bacterial flagella. Annu Rev Genet 1992;26:131. Reproduced with permission from Annual Review of Genetics, Volume 26, © 1992 by Annual Reviews.)
Flagella are made stepwise (see Figure 2-23). First, the basal body is assembled and inserted into the cell envelope. Then the hook is added, and finally, the filament is assembled progressively by the addition of flagellin subunits to its growing tip. The flagellin subunits are extruded through a hollow central channel in the flagella; when it reaches the tip, it condenses with its predecessors, and thus the filament elongates.
Bacterial flagella are semirigid helical rotors to which the cell imparts a spinning movement. Rotation is driven by the flow of protons into the cell down the gradient produced by the primary proton pump (see earlier discussion); in the absence of a metabolic energy source, it can be driven by a proton motive force generated by ionophores. Bacteria living in alkaline environments (alkalophiles) use the energy of the sodium ion gradient—rather than the proton gradient—to drive the flagellar motor (Figure 2-24).
Structural components within the basal body of the flagellum allow the inner portion of this structure, the rods of the basal body, and the attached hook–filament complex to rotate. The outer rings remain statically in contact with the inner and outer cell membranes and cell wall (murein), anchoring the flagellum complex to the bacterial cell envelope. Rotation is driven by the flow of protons through the motor from the periplasmic space, outside the cell membrane, into the cytoplasm in response to the electric field and proton gradient across the membrane, which together constitute the proton motive force. A switch determines the direction of rotation, which in turn determines whether the bacteria swim forward (by counterclockwise rotation of the flagellum) or tumble (caused by clockwise rotation of the flagellum). (Reproduced with permission from Saier MH Jr: Peter Mitchell and his chemiosmotic theories. ASM News 1997;63:13.)
All the components of the flagellar motor are located in the cell envelope. Flagella attached to isolated, sealed cell envelopes rotate normally when the medium contains a suitable substrate for respiration or when a proton gradient is artificially established.
When a peritrichous bacterium swims, its flagella associate to form a posterior bundle that drives the cell forward in a straight line by counterclockwise rotation. At intervals, the flagella reverse their direction of rotation and momentarily dissociate, causing the cell to tumble until swimming resumes in a new, randomly determined direction. This behavior makes possible the property of chemotaxis: A cell that is moving away from the source of a chemical attractant tumbles and reorients itself more frequently than one that is moving toward the attractant, the result being the net movement of the cell toward the source. The presence of a chemical attractant (eg, a sugar or an amino acid) is sensed by specific receptors located in the cell membrane (in many cases, the same receptor also participates in membrane transport of that molecule). The bacterial cell is too small to be able to detect the existence of a spatial chemical gradient (ie, a gradient between its two poles); rather, experiments show that it detects temporal gradients, that is, concentrations that decrease with time during which the cell is moving away from the attractant source and increase with time during which the cell is moving toward it.
Some compounds act as repellants rather than attractants. One mechanism by which cells respond to attractants and repellents involves a cGMP-mediated methylation and demethylation of specific proteins in the membrane. Whereas attractants cause a transient inhibition of demethylation of these proteins, repellents stimulate their demethylation.
The mechanism by which a change in cell behavior is brought about in response to a change in the environment is called sensory transduction. Sensory transduction is responsible not only for chemotaxis but also for aerotaxis (movement toward the optimal oxygen concentration), phototaxis (movement of photosynthetic bacteria toward the light), and electron acceptor taxis (movement of respiratory bacteria toward alternative electron acceptors, such as nitrate and fumarate). In these three responses, as in chemotaxis, net movement is determined by regulation of the tumbling response.
Many gram-negative bacteria possess rigid surface appendages called pili (L “hairs”) or fimbriae (L “fringes”). They are shorter and finer than flagella; similar to flagella, they are composed of structural protein subunits termed pilins. Some pili contain a single type of pilin, others more than one. Minor proteins termed adhesins are located at the tips of pili and are responsible for the attachment properties. Two classes can be distinguished: ordinary pili, which play a role in the adherence of symbiotic and pathogenic bacteria to host cells; and sex pili, which are responsible for the attachment of donor and recipient cells in bacterial conjugation (see Chapter 7). Pili are illustrated in Figure 2-25, in which the sex pili have been coated with phage particles for which they serve as specific receptors.
Pili. Pili on an Escherichia coli cell. The short pili (fimbriae) mediate adherence; the sex pilus is involved in DNA transfer. (Courtesy of Dr. Charles Brinton, Jr.)
Motility via pili is completely different from flagellar motion. Pilin molecules are arranged helically to form a straight cylinder that does not rotate and lacks a complete basal body. Their tips strongly adhere to surfaces at a distance from the cells. Pili then depolymerize from the inner end, thus retracting inside the cell. The result is that the bacterium moves in the direction of the adhering tip. This kind of surface motility is called twitching and is widespread among piliated bacteria. Unlike flagella, pili grow from the inside of the cell outward.
The virulence of certain pathogenic bacteria depends on the production not only of toxins but also of “colonization antigens,” which are ordinary pili that provide the cells with adherent properties. In enteropathogenic E coli strains, both the enterotoxins and the colonization antigens (pili) are genetically determined by transmissible plasmids, as discussed in Chapter 7.
In one group of gram-positive cocci, the streptococci, fimbriae are the site of the main surface antigen, the M protein. Lipoteichoic acid, associated with these fimbriae, is responsible for the adherence of group A streptococci to epithelial cells of their hosts.
Pili of different bacteria are antigenically distinct and elicit the formation of antibodies by the host. Antibodies against the pili of one bacterial species will not prevent the attachment of another species. Some bacteria (see Chapter 21), such as N gonorrhoeae, are able to make pili of different antigenic types (antigenic variation) and thus can still adhere to cells in the presence of antibodies to their original type of pili. Similar to capsules, pili inhibit the phagocytic ability of leukocytes.
Members of several bacterial genera are capable of forming endospores (Figure 2-26). The two most common are gram-positive rods: the obligately aerobic genus Bacillus and the obligately anaerobic genus Clostridium. The other bacteria known to form endospores are Thermoactinomyces, Sporolactobacillus, Sporosarcina, Sporotomaculum, Sporomusa, and Sporohalobacter spp. These organisms undergo a cycle of differentiation in response to environmental conditions: The process, sporulation, is triggered by near depletion of any of several nutrients (carbon, nitrogen, or phosphorous). Each cell forms a single internal spore that is liberated when the mother cell undergoes autolysis. The spore is a resting cell, highly resistant to desiccation, heat, and chemical agents; when returned to favorable nutritional conditions and activated (see below), the spore germinates to produce a single vegetative cell.
Sporulating cells of bacillus species. A: Unidentified bacillus from soil. B: Bacillus cereus. C: Bacillus megaterium. (Reproduced with permission from Robinow CF: Structure. In Gunsalus IC, Stanier RY [editors]. The Bacteria: A Treatise on Structure and Function, Vol 1. Academic Press, 1960.)
The sporulation process begins when nutritional conditions become unfavorable, near depletion of the nitrogen or carbon source (or both) being the most significant factor. Sporulation occurs massively in cultures that have terminated exponential growth as a result of this near depletion.
Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components. These changes represent a true process of differentiation: A series of genes whose products determine the formation and final composition of the spore are activated. These changes involve alterations in the transcriptional specificity of RNA polymerase, which is determined by the association of the polymerase core protein with one or another promoter-specific protein called a sigma factor. During vegetative growth, a sigma factor designated σA predominates. Then, during sporulation, five other sigma factors are formed that cause various spore genes to be expressed at various times in specific locations.
The sequence of events in sporulation is highly complex: Differentiation of a vegetative cell of B subtilis into an endospore takes about 7 hours under laboratory conditions. Different morphologic and chemical events occur at sequential stages of the process. Seven different stages have been identified.
Morphologically, sporulation begins with the formation of an axial filament (Figure 2-27). The process continues with an infolding of the membrane so as to produce a double-membrane structure whose facing surfaces correspond to the cell wall–synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.
The stages of endospore formation. (Reproduced with permission from Merrick MJ: Streptomyces. In: Parish JH [editor]. Developmental Biology of Procaryotes. Univ California Press, 1979.)
The two spore membranes now engage in the active synthesis of special layers that will form the cell envelope: the spore wall and the cortex, lying outside the facing membranes. In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents.
B. Properties of Endospores
1. Core—The core is the spore protoplast. It contains a complete nucleus (chromosome), all of the components of the protein-synthesizing apparatus, and an energy-generating system based on glycolysis. Cytochromes are lacking even in aerobic species, the spores of which rely on a shortened electron transport pathway involving flavoproteins. A number of vegetative cell enzymes are increased in amount (eg, alanine racemase), and a number of unique enzymes are formed (eg, dipicolinic acid synthetase). Spores contain no reduced pyridine nucleotides or ATP. The energy for germination is stored as 3-phosphoglycerate rather than as ATP.
The heat resistance of spores is partly attributable to their dehydrated state and in part to the presence in the core of large amounts (5–15% of the spore dry weight) of calcium dipicolinate, which is formed from an intermediate of the lysine biosynthetic pathway (see Figure 6-19). In some way not yet understood, these properties result in the stabilization of the spore enzymes, most of which exhibit normal heat lability when isolated in soluble form.
2. Spore wall—The innermost layer surrounding the inner spore membrane is called the spore wall. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.
3. Cortex—The cortex is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan, with many fewer cross-links than are found in cell wall peptidoglycan. Cortex peptidoglycan is extremely sensitive to lysozyme, and its autolysis plays a role in spore germination.
4. Coat—The coat is composed of a keratin-like protein containing many intramolecular disulfide bonds. The impermeability of this layer confers on spores their relative resistance to antibacterial chemical agents.
5. Exosporium—The exosporium is composed of proteins, lipids, and carbohydrates. It consists of a paracrystalline basal layer and a hairlike outer region. The function of the exosporium is unclear. Spores of some Bacillus species (eg, B anthracis and B cereus) possess an exosporium, but other species (eg, B atrophaeus) have spores that lack this structure.
The germination process occurs in three stages: activation, initiation, and outgrowth.
1. Activation—Most endospores cannot germinate immediately after they have formed. But they can germinate after they have rested for several days or are first activated in a nutritionally rich medium by one or another agent that damages the spore coat. Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and compounds containing free sulfhydryl groups.
2. Initiation—After activation, a spore will initiate germination if the environmental conditions are favorable. Different species have evolved receptors that recognize different effectors as signaling a rich medium: Thus, initiation is triggered by l-alanine in one species and by adenosine in another. Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.
3. Outgrowth—Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. A period of active biosynthesis follows; this period, which terminates in cell division, is called outgrowth. Outgrowth requires a supply of all nutrients essential for cell growth.