Gram-negative cell walls contain three components that lie outside of the peptidoglycan layer: outer membrane, lipopolysaccharide, and lipoprotein (Figure1).

Fig1. 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 E. 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 cytoplasmic membrane, and its outer leaf let contains a distinctive component, a lipopolysaccharide (LPS) (see next). As a result, this is an asymmetrical mem brane, 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 mem branes 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 resistance of Gram-negative bacteria to some antibiotics. 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 the inner and outer leaflets of the outer membrane (Figure 2). They form relatively nonspecific pores that permit the free diffusion of small hydrophilic solutes across the outer 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.

Fig2. 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.)

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.

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 prob ably 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 mem brane, based on cross-linking studies and analyses of functional relationships, is shown in Figure 2-19. 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. 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. They are translocated into the periplasm via the Sec translocase. They then fold in the periplasm before being inserted into the outer membrane. In E. coli, YaeT appears to function primarily in outer membrane protein insertion.

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 3A). 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. In E. coli, LPS insertion is mediated by OstA. The presence of LPS is required for the function of many outer membrane proteins.

Fig3. 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 E. 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 JM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill; 2008. © McGraw-Hill Education.)

Lipid A consists of phosphorylated glucosamine disaccharide units to which are attached several long-chain fatty acids (Figure 3). 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 3A 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 3A. 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 mem brane permeable to large hydrophobic molecules.

LPS, 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 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 com posed of a variable number of repeated oligosaccharide units (see Figure 3); 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, the structures of these glycolipids 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 organ isms 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 1). The lipoprotein contains 57 amino acids, rep resenting 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 trans portable 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 oligo saccharides appear to play a role in osmoregulation because cells grown in media of low osmolarity increase their synthesis of these compounds 16-fold.

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مواضيع ذات صلة

The Acid-Fast Cell Wall

Special Components of Gram-Positive Cell Walls

The Peptidoglycan Layer

Bacterial Cultivation

Principles of Bacterial Cultivation : Environmental Requirements

Structure and Function of the Bacterial Cell

Bacterial Metabolism

Bacterial Genetics: Nucleic Acid Structure and Organization

Detection of Bacteremia: Culture Techniques

Detection of Bacteremia: Types of Blood Culture Bottle

Detection of Bacteremia: Specimen Collection

The Study of the Intestinal Microbiota