Of special interest as a component of the Gram-negative cell wall is the outer membrane, a discrete bilayered structure on the outside of the peptidoglycan sheet (see Figure 18 below). For the bacterium, the outer membrane is first and foremost a permeability barrier, but primarily due to its lipopolysaccharide content, it possesses many interesting and important characteristics of Gram-negative bacteria. The outer membrane is a lipid bilayer intercalated with proteins, superficially resembling the plasma membrane. The inner face of the outer membrane is composed of phospholipids similar to the phosphoglycerides that compose the plasma membrane. The outer face of the outer membrane may contain some phospholipid, but mainly it is formed by a different type of amphiphilic molecule which is composed of lipopolysaccharide (LPS). Outer membrane proteins usually traverse the membrane and in one case, anchor the outer membrane to the underlying peptidoglycan sheet.

Figure 18. Schematic illustration of the outer membrane, cell wall and plasma membrane of a Gram-negative bacterium. Note the structure and arrangement of molecules that constitute the outer membrane.

The LPS molecule that constitutes the outer face of the outer membrane is composed of a hydrophobic region, called Lipid A, that is attached to a hydrophilic linear polysaccharide region, consisting of the core polysaccharide and the O-specific polysaccharide.

Figure 19. Structure of LPS

The Lipid A head of the molecule inserts into the interior of the membrane, and the polysaccharide tail of the molecule faces the aqueous environment. Where the tail of the molecule inserts into the head there is an accumulation of negative charges such that a magnesium cation is chelated between adjacent LPS molecules. This provides the lateral stability for the outer membrane, and explains why treatment of Gram-negative bacteria with a powerful chelating agent, such as EDTA, causes dispersion of LPS molecules.

Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts LPS or endotoxin activates macrophages to produce pyrogens, activates the complement cascade causing inflammation, and activates blood factors resulting in intravascular coagulation and hemorrhage. Endotoxins may play a role in infection by any Gram-negative bacterium. The toxic component of endotoxin (LPS) is Lipid A. The O-specific polysaccharide may provide ligands for bacterial attachment and confer some resistance to phagocytosis. Variation in the exact sugar content of the O polysaccharide (also referred to as the O antigen) accounts for multiple antigenic types (serotypes) among Gram-negative bacterial pathogens. Therefore. even though Lipid A is the toxic component in LPS, the polysaccharides nonetheless contribute to virulence of Gram-negative bacteria.

The proteins in the outer membrane of Escherichia coli are well characterized (see Table 5). About 400,00 copies of the Braun lipoprotein are covalently attached to the peptidoglycan sheet at one end and inserted into the hydrophobic interior of the membrane at the opposite end. A group of trimeric proteins called porins form pores of a fixed diameter through the lipid bilayer of the membrane. The omp C and omp F porins of E. coli are designed to allow passage of hydrophilic molecules up to mw of about 750 daltons. Larger molecules or harmful hydrophobic compounds (such as bile salts in the intestinal tract) are excluded from entry. Porins are designed in Gram-negative bacteria to allow passage of useful molecules (nutrients) through the barrier of the outer membrane, but to exclude passage harmful substances from the environment. The ubiquitous omp A protein in the outer membrane of E. coli has a porin like structure, and may function in uptake of specific ions, but it is also a receptor for the F pilus and an attachment site for bacterial viruses.

Table 5. Functions of the outer membrane components of Escherichia coli.

Component	Function
Lipopolysaccharide (LPS)	Permeability barrier
Mg++ bridges	Stabilizes LPS and is essential for its permeability characteristics
Braun lipoprotein	Anchors the outer membrane to peptidoglycan (murein) sheet
Omp C and Omp F porins	proteins that form pores or channels through outer membrane for passage of hydrophilic molecules
Omp A protein	provides receptor for some viruses and bacteriocins; stabilizes mating cells during conjugation

S-layers

S-layer proteins form the outermost cell envelope component of a broad spectrum of bacteria and archaea.  S-layers are composed of a single protein or glycoprotein species (Mw 40-200 kDa) and exhibit either oblique, square or hexagonal lattice symmetry with unit cell dimensions in the range of 3 to 30 nm. S-layers are generally 5 to 10 nm thick and show pores of identical size (diameter, 2 - 8 nm) and morphology.

Crystalline bacterial cell surface layer (S-layer) proteins have been optimized during billions of years of biological evolution as constituent elements of one of the simplest self-assembly systems in nature. Isolated S-layer proteins have the intrinsic property to recrystallize into two-dimensional arrays on a broad spectrum of surfaces including silicon, metals and polymers, and to interfaces such as planar lipid films and liposomes. The well defined arrangement of functional groups on S-layer lattices allows the binding of molecules and particles in defined regular arrays. S-layers also represent templates for the formation of inorganic nanocrystal superlattices composed of CdS, Au, Ni, Pt, or Pd.

The self-assembly of S-layers illustrates a basic building principle in nature for generating large arrays of biomolecules with well-defined geometrical and physicochemical surface properties.

The S-layer can function as an adhesin, enabling the bacterium to adhere to host cell membranes and environmental surfaces in order to colonize. Many of the cell-associated protein adhesins used by pathogens are components of the S-layer.

A correlation between Gram stain reaction and cell wall properties of bacteria is summarized in Table 6. The Gram stain procedure contains a "destaining" step wherein the cells are washed with an acetone-alcohol mixture. The lipid content of the Gram-negative wall probably affects the outcome of this step so that Gram-positive cells retain a primary stain while Gram-negative cells are destained.

Table 6. Correlation of Grams stain with other properties of Bacteria.

Property	Gram-positive	Gram-negative
Thickness of wall	thick (20-80 nm)	thin (10 nm)
Number of layers	1	2
Peptidoglycan (murein) content	>50%	10-20%
Teichoic acids in wall	present	absent
Lipid and lipoprotein content	0-3%	58%
Protein content	0	9%
Lipopolysaccharide content	0	13%
Sensitivity to Penicillin G	yes	no (1)
Sensitivity to lysozyme	yes	no (2)

(1) A few Gram-negative bacteria are sensitive to natural penicillins. Many Gram-negative bacteria are sensitive to some type of penicillin, especially semisynthetic penicillins. Gram-negative bacteria, including E. coli, can be made sensitive to natural penicillin by procedures that disrupt the permeability characteristics of the outer membrane.
(2) Gram-negative bacteria are sensitive to lysozyme if pretreated by some procedure that removes the outer membrane and exposes the peptidoglycan directly to the enzyme.