Figure 2-5
(A) Electron micrograph of a thin section of the Gram-positive M. lysodeikticus showing the thick peptidoglycan cell wall (cw), underlying cytoplasmic (plasma) membrane (cm), mesosome (m), and nucleus (n). (B) Freeze-fractured Bacteriodes cell showing typical major convex fracture faces through the inner (im) and outer (om) membranes. Bars = 1 µm; circled arrow in B indicates direction of shadowing.
Some bacteria form capsules, which constitute the outermost layer of the bacterial cell and surround it with a relatively thick layer of viscous gel. Capsules may be up to 10 µm thick. Some organisms lack a well-defined capsule but have loose, amorphous slime layers external to the cell wall or cell envelope. The α hemolytic Streptococcus mutans, the primary organism found in dental plaque is able to synthesis a large extracellular mucoid glucans from sucrose. Not all bacterial species produce capsules; however, the capsules of encapsulated pathogens are often important determinants of virulence. Encapsulated species are found among both Gram-positive and Gram-negative bacteria. In both groups, most capsules are composed of highmolecular-weight viscous polysaccharides that are retained as a thick gel outside the cell wall or envelope. The capsule of Bacillus anthracis (the causal agent of anthrax) is unusual in that it is composed of a γ-glutamyl polypeptide. Table 2-1 presents the various capsular substances formed by a selection of Gram-positive and Gram-negative bacteria. A plasma membrane stage is involved in the biosynthesis and assembly of the capsular substances, which are extruded or secreted through the outer wall or envelope structures. Mutational loss of enzymes involved in the biosynthesis of the capsular polysaccharides can result in the smooth-to-rough variation seen in the pneumococci.
The capsule is not essential for viability. Viability is not affected when capsular polysaccharides are removed enzymatically from the cell surface. The exact functions of capsules are not fully understood, but they do confer resistance to phagocytosis and hence provide the bacterial cell with protection against host defenses to invasion.
Table 2-1
The Gram stain broadly differentiates bacteria into Gram-positive and Gram-negative groups; a few organisms are consistently Gram-variable. Gram-positive and Gram-negative organisms differ drastically in the organization of the structures outside the plasma membrane but below the capsule (Fig. 2-6): in Gram-negative organisms these structures constitute the cell envelope, whereas in Gram-positive organisms they are called a cell wall.
Most Gram-positive bacteria have a relatively thick (about 20 to 80 nm), continuous cell wall (often called the sacculus), which is composed largely of peptidoglycan (also known as mucopeptide or murein). In thick cell walls, other cell wall polymers (such as the teichoic acids, polysaccharides, and peptidoglycolipids) are covalently attached to the peptidoglycan. In contrast, the peptidoglycan layer in Gram-negative bacteria is thin (about 5 to 10 nm thick); in E. coli, the peptidoglycan is probably only a monolayer thick. Outside the peptidoglycan layer in the Gram-negative envelope is an outer membrane structure (about 7.5 to 10 nm thick). In most Gram-negative bacteria, this membrane structure is anchored noncovalently to lipoprotein molecules (Braun's lipoprotein), which, in turn, are covalently linked to the peptidoglycan. The lipopolysaccharides of the Gram-negative cell envelope form part of the outer leaflet of the outer membrane structure.
The organization and overall dimensions of the outer membrane of the Gram-negative cell envelope are similar to those of the plasma membrane (about 7.5 nm thick). Moreover, in Gram-negative bacteria such as E. coli, the outer and inner membranes adhere to each other at several hundred sites (Bayer patches); these sites can break up the continuity of the peptidoglycan layer. Table 2-2 summarizes the major classes of chemical constituents in the walls and envelopes of Gram-positive and Gram-negative bacteria.
The basic differences in surface structures of Gram-positive and Gram-negative bacteria explain the results of Gram staining. Both Gram-positive and Gram-negative bacteria take up the same amounts of crystal violet (CV) and iodine (I). The CV-I complex, however, is trapped inside the Gram-positive cell by the dehydration and reduced porosity of the thick cell wall as a result of the differential washing step with 95 percent ethanol or other solvent mixture. In contrast, the thin peptidoglycan layer and probable discontinuities at the membrane adhesion sites do not impede solvent extraction of the CV-I complex from the Gram-negative cell. The above mechanism of the Gram stain based on the structural differences between the two groups has been confirmed by sophisticated methods of electron microscopy (see Ref. Bereridge and Daries). The sequence of steps in the Gram stain differentiation is illustrated diagrammatically in Figure 2-7. Moreover, mechanical disruption of the cell wall of Gram-positive organisms or its enzymatic removal with lysozyme results in complete extraction of the CV-I complex and conversion to a Gram-negative reaction. Therefore, autolytic wall-degrading enzymes that cause cell wall breakage may account for Gram-negative or variable reactions in cultures of Gram-positive organisms (such as Staphylococcus aureus, Clostridium perfringens, Corynebacterium diphtheriae, and some Bacillus spp.).
Figure 2-6
Note the complexity of the Gram-negative cell envelope (outer membrane, its hydrophobic lipoprotein anchor; periplasmic space).
The organization and overall dimensions of the outer membrane of the Gram-negative cell envelope are similar to those of the plasma membrane (about 7.5 nm thick). Moreover, in Gram-negative bacteria such as E. coli, the outer and inner membranes adhere to each other at several hundred sites (Bayer patches); these sites can break up the continuity of the peptidoglycan layer. Table 2-2 summarizes the major classes of chemical constituents in the walls and envelopes of Gram-positive and Gram-negative bacteria.
Table 2-2
Figure 2-7
Unique features of almost all prokaryotic cells (except for Halobacterium halobium and mycoplasmas) are cell wall peptidoglycan and the specific enzymes involved in its biosynthesis. These enzymes are target sites for inhibition of peptidoglycan synthesis by specific antibiotics. The primary chemical structures of peptidoglycans of both Gram-positive and Gram-negative bacteria have been established; they consist of a glycan backbone of repeating groups of β1, 4-linked disaccharides of β1,4-N-acetylmuramyl-N-acetylglucosamine. Tetrapeptides of L-alanine-D-isoglutamic acid-L-lysine (or diaminopimelic acid)-n-alanine are linked through the carboxyl group by amide linkage of muramic acid residues of the glycan chains; the D-alanine residues are directly cross-linked to the 𝛆-amino group of lysine or diaminopimelic acid on a neighboring tetrapeptide, or they are linked by a peptide bridge. In S. aureus peptidoglycan, a glycine pentapeptide bridge links the two adjacent peptide structures. The extent of direct or peptide-bridge cross-linking varies from one peptidoglycan to another. The staphylococcal peptidoglycan is highly cross-linked, whereas that of E. coli is much less so, and has a more open peptidoglycan mesh. The diamino acid providing the 𝛆-amino group for cross-linking is lysine or diaminopimelic acid, the latter being uniformly present in Gram-negative peptidoglycans. The structure of the peptidoglycan is illustrated in Figure 2-8. A peptidoglycan with a chemical structure substantially different from that of all eubacteria has been discovered in certain archaebacteria. Instead of muramic acid, this peptidoglycan contains talosaminuronic acid and lacks the D-amino acids found in the eubacterial peptidoglycans. Interestingly, organisms containing this wall polymer (referred to as pseudomurein) are insensitive to penicillin, an inhibitor of the transpeptidases involved in peptidoglycan biosynthesis in eubacteria.
The ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine is specifically cleaved by the bacteriolytic enzyme lysozyme. Widely distributed in nature, this enzyme is present in human tissues and secretions and can cause complete digestion of the peptidoglycan walls of sensitive organisms. When lysozyme is allowed to digest the cell wall of Gram-positive bacteria suspended in an osmotic stabilizer (such as sucrose), protoplasts are formed. These protoplasts are able to survive and continue to grow on suitable media in the wall-less state. Gram-negative bacteria treated similarly produce spheroplasts, which retain much of the outer membrane structure. The dependence of bacterial shape on the peptidoglycan is shown by the transformation of rod-shaped bacteria to spherical protoplasts (spheroplasts) after enzymatic breakdown of the peptidoglycan. The mechanical protection afforded by the wall peptidoglycan layer is evident in the osmotic fragility of both protoplasts and spheroplasts. There are two groups of bacteria that lack the protective cell wall peptidoglycan structure, the Mycoplasma species, one of which causes atypical pneumonia and some genitourinary tract infections and the L-forms, which originate from Gram-positive or Gram-negative bacteria and are so designated because of their discovery and description at the Lister Institute, London. The mycoplasmas and L-forms are all Gram-negative and insensitive to penicillin and are bounded by a surface membrane structure. L-forms arising "spontaneously" in cultures or isolated from infections are structurally related to protoplasts and spheroplasts; all three forms (protoplasts, spheroplasts, and L-forms) revert infrequently and only under special conditions.
Figure 2-8
Wall teichoic acids are found only in certain Gram-positive bacteria (such as staphylococci, streptococci, lactobacilli, and Bacillus spp.); so far, they have not been found in gram- negative organisms. Teichoic acids are polyol phosphate polymers, with either ribitol or glycerol linked by phosphodiester bonds; their structures are illustrated in Figure 2-9. Substituent groups on the polyol chains can include D-alanine (ester linked), N-acetylglucosamine, N-acetylgalactosamine, and glucose; the substituent is characteristic for the teichoic acid from a particular bacterial species and can act as a specific antigenic determinant. Teichoic acids are covalently linked to the peptidoglycan. These highly negatively charged polymers of the bacterial wall can serve as a cation-sequestering mechanism.
Figure 2-9
(A) Ribitol teichoic acid with repeating units of 1,5-phosphodiester linkages of D-ribitol and D-alanyl ester on position 2 and glycosyl substituents (R) on position 4. The glycosyl groups may abe N-acetylglucosaminyl (α or β) as in S. aureus or α-glucosyl as in B. subtilis W23. (B) Glycerol teichoic acid with 1,3-phosphodiester linkages of glycerol repeating units (1,2-linkages in some species). In the glycerol teichoic acid structure shown, the polymer may be unsubstituted (R - H) or substituted (R - D-alanyl or glycosyl).
In addition to the principal cell wall polymers, the walls of certain Gram-positive bacteria possess polysaccharide molecules linked to the peptidoglycan. For example, the C polysaccharide of streptococci confers group specificity. Acidic polysaccharides attached to the peptidoglycan are called teichuronic acids. Mycobacteria have peptidoglycolipids, glycolipids, and waxes associated with the cell wall.
A characteristic feature of Gram-negative bacteria is possession of various types of complex macromolecular lipopolysaccharide (LPS). So far, only one Gram-positive organism, Listeria monocytogenes, has been found to contain an authentic LPS. The LPS of this bacterium and those of all Gram-negative species are also called endotoxins, thereby distinguishing these cell-bound, heat-stable toxins from heat-labile, protein exotoxins secreted into culture media. Endotoxins possess an array of powerful biologic activities and play an important role in the pathogenesis of many Gram-negative bacterial infections. In addition to causing endotoxic shock, LPS is pyrogenic, can activate macrophages and complement, is mitogenic for B lymphocytes, induces interferon production, causes tissue necrosis and tumor regression, and has adjuvant properties. The endotoxic properties of LPS reside largely in the lipid A components. Usually, the LPS molecules have three regions: the lipid A structure required for insertion in the outer leaflet of the outer membrane bilayer; a covalently attached core composed of 2-keto-3deoxyoctonic acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose, and galactose; and polysaccharide chains linked to the core. The polysaccharide chains constitute the O-antigens of the Gram-negative bacteria, and the individual monosaccharide constituents confer serologic specificity on these components. Figure 2-10 depicts the structure of LPS. Although it has been known that lipid A is composed of β1,6-linked D-glucosamine disaccharide substituted with phosphomonester groups at positions 4' and 1, uncertainties have existed about the attachment positions of the six fatty acid acyl and KDO groups on the disaccharide. The demonstration of the structure of lipid A of LPS of a heptoseless mutant of Salmonella typhimurium has established that amide-linked hydroxymyristoyl and lauroxymyristoyl groups are attached to the nitrogen of the 2- and 2'-carbons, respectively, and that hydroxymyristoyl and myristoxymyristoyl groups are attached to the oxygen of the 3- and 3'-carbons of the disaccharide, respectively. Therefore, only position 6' is left for attachment of KDO units.
LPS and phospholipids help confer asymmetry to the outer membrane of the Gram-negative bacteria, with the hydrophilic polysaccharide chains outermost. Each LPS is held in the outer membrane by relatively weak cohesive forces (ionic and hydrophobic interactions) and can be dissociated from the cell surface with surface-active agents.
As in peptidoglycan biosynthesis, LPS molecules are assembled at the plasma or inner membrane. These newly formed molecules are initially inserted into the outer-inner membrane adhesion sites.
Figure 2-10
As in peptidoglycan biosynthesis, LPS molecules are assembled at the plasma or inner membrane. These newly formed molecules are initially inserted into the outer-inner membrane adhesion sites.
In thin sections, the outer membranes of Gram-negative bacteria appear broadly similar to the plasma or inner membranes; however, they differ from the inner membranes and walls of Gram-positive bacteria in numerous respects. The lipid A of LPS is inserted with phospholipids to create the outer leaflet of the bilayer structure; the lipid portion of the lipoprotein and phospholipid form the inner leaflet of the outer membrane bilayer of most Gram-negative bacteria (Fig. 2-6).
In addition to these components, the outer membrane possesses several major outer membrane proteins; the most abundant is called porin. The assembled subunits of porin form a channel that limits the passage of hydrophilic molecules across the outer membrane barrier to those having molecular weights that are usually less than 600 to 700. Evidence also suggests that hydrophobic pathways exist across the outer membrane and are partly responsible for the differential penetration and effectiveness of certain b-lactam antibiotics (ampicillin, cephalosporins) that are active against various Gram-negative bacteria. Although the outer membranes act as a permeability barrier or molecular sieve, they do not appear to possess energy-transducing systems to drive active transport. Several outer membrane proteins, however, are involved in the specific uptake of metabolites (maltose, vitamin B12, nucleosides) and iron from the medium. Thus, outer membranes of the Gram-negative bacteria provide a selective barrier to external molecules and thereby prevent the loss of metabolite-binding proteins and hydrolytic enzymes (nucleases, alkaline phosphatase) found in the periplasmic space. The periplasmic space is the region between the outer surface of the inner (plasma) membrane and the inner surface of the outer membrane (Figure 2-6). Thus, Gram-negative bacteria have a cellular compartment that has no equivalent in Gram-positive organisms. In addition to the hydrolytic enzymes, the periplasmic space holds binding proteins (proteins that specifically bind sugars, amino acids, and inorganic ions) involved in membrane transport and chemotactic receptor activities. Moreover, plasmid-encoded b-lactamases and aminoglycoside-modifying enzymes (phosphorylation or adenylation) in the periplasmic space produce antibiotic resistance by degrading or modifying an antibiotic in transit to its target sites on the membrane (penicillin-binding proteins) or on the ribosomes (aminoglycosides). These periplasmic proteins can be released by subjecting the cells to osmotic shock and after treatment with the chelating agent ethylenediaminetetraacetic acid.
In addition to these components, the outer membrane possesses several major outer membrane proteins; the most abundant is called porin. The assembled subunits of porin form a channel that limits the passage of hydrophilic molecules across the outer membrane barrier to those having molecular weights that are usually less than 600 to 700. Evidence also suggests that hydrophobic pathways exist across the outer membrane and are partly responsible for the differential penetration and effectiveness of certain b-lactam antibiotics (ampicillin, cephalosporins) that are active against various Gram-negative bacteria. Although the outer membranes act as a permeability barrier or molecular sieve, they do not appear to possess energy-transducing systems to drive active transport. Several outer membrane proteins, however, are involved in the specific uptake of metabolites (maltose, vitamin B12, nucleosides) and iron from the medium. Thus, outer membranes of the Gram-negative bacteria provide a selective barrier to external molecules and thereby prevent the loss of metabolite-binding proteins and hydrolytic enzymes (nucleases, alkaline phosphatase) found in the periplasmic space. The periplasmic space is the region between the outer surface of the inner (plasma) membrane and the inner surface of the outer membrane (Figure 2-6). Thus, Gram-negative bacteria have a cellular compartment that has no equivalent in Gram-positive organisms. In addition to the hydrolytic enzymes, the periplasmic space holds binding proteins (proteins that specifically bind sugars, amino acids, and inorganic ions) involved in membrane transport and chemotactic receptor activities. Moreover, plasmid-encoded b-lactamases and aminoglycoside-modifying enzymes (phosphorylation or adenylation) in the periplasmic space produce antibiotic resistance by degrading or modifying an antibiotic in transit to its target sites on the membrane (penicillin-binding proteins) or on the ribosomes (aminoglycosides). These periplasmic proteins can be released by subjecting the cells to osmotic shock and after treatment with the chelating agent ethylenediaminetetraacetic acid.