Details information on Cell Wall of Gram-negative bacteria

Gram-negative bacteria are a class of bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation, making positive identification possible. The thin peptidoglycan layer of their cell wall is sandwiched between an inner cell membrane and a bacterial outer membrane. In Gram staining, the outer lipid-based membrane of gram-negative bacteria is removed by an alcohol solution. The alcohol also decolorizes the then exposed peptidoglycan layer by dissolving away the previously applied crystal violet. A counterstain (safranin or fuchsine) is then added which recolorizes the bacteria red or pink.

Characteristics

Gram-negative bacteria display the following characteristics:

1. Cell membrane (cytoplasmic).
2. Thin peptidoglycan layer (which is much thicker in gram-positive bacteria).
3. Outer membrane containing lipopolysaccharide (LPS, which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet.
4. Porins exist in the outer membrane, which act like pores for particular molecules.
5. There is a space between the peptidoglycan layer and the secondary cell membrane called the periplasmic space.
6. The S-layer is directly attached to the outer membrane rather than the peptidoglycan.
7. If present, flagella have four supporting rings instead of two.
8. No teichoic acids or lipoteichoic acids are present.
9. Lipoproteins are attached to the polysaccharide backbone.
10. Some of them contain Braun's lipoprotein, which serves as a link between the outer membrane and the peptidoglycan chain by a covalent bond.
11. Most, with very few exceptions, do not form spores.
12. Release some endotoxin.

Cell Wall

The cell wall is a rigid structure that surrounds the bacterial cell just outside of the plasma membrane.

Functions to:

• Gives the bacterium its shape

• Gives mechanical strength to the cell and protects it from exploding due to osmotic lysis. (shape and strength due to the peptidoglycan) 

Gram Negative Cell Wall

• The Gram Negative cell wall has a peptidoglycan layer that is about 2-3 nm thick (thin compared to the gram positive) and makes up about 5-20% of the dry weight of the cell.

• There is an outer membrane than lies outside of the thin peptidoglycan layer.

Braun’s Lipoprotein:

• Most abundant membrane protein.

• Covalently joined to the peptidoglycan and embedded in the the outer membrane.

• The peptidogylcan and the outer membrane are firmly linked by this protein.

 Adhesion Site:

• Strengthens the wall and holds the outer membrane in place.

• Regions of direct contact or possibly true membrane fusions.

• Proposed that substances can move into the cell through these adhesion sites rather than through the periplasm. 

Peptidoglycan Layer:

• The peptidoglycan layer is covered by an outer membrane that contains various proteins as well as lipopolysaccharides (LPS).

• Lipopolysaccharides (LPS) is a large complex molecule containing both lipids and carbohydrates.

LPS consists of three parts: 

Lipid A

• Consists of two glucosamine sugar derivitives each attached to three fatty acids and phosphate.

• The lipid A residue is buried  within the outer membrane and the remaining components are projected from the cell surface.

Core Polysaccharide

 The core polysaccharide consists of 10 sugar residues, most of which have an unusual structure, and are attached to the lipid A moiety.

O-Side Chain

The LPS structure ends with a terminal O side chain.  The O antigen is a short polysaccharide chain that varies in composition and generally contains a number of unusual sugar residues.

LPS Importance

·         The LPS is negatively charged an contributes to the overall negative charge of the cell (like the teichoic acids of Gm+ cells)

·         More importantly, the Lipid A molecule is toxic and serves as a super-antigen to induce an inflammitory immune response (endotoxin).

·         (Teichoic acids are the major antigens of Gm+ cells, LPS is the major antigen of GM- cells)

Outer Membrane Function:

• The outer membrane functions as a permeability barrier that protects the cell from a number of toxic agents, including some dyes and several antibiotics

• Outer membrane is more permeable than the plasma membrane and permits the passage of small molecules like glucose, due to porins.

• The outer membrane contains a number of protein channels called porins that make the lipid bilayer relatively permeable to small molecules.  Three porins molecules band together span the membrane to form narrow channels through which smaller molecules can pass.

 S-Layers

 Functions:

• May protect cell against ion  and pH fluctuations, osmotic stress, enzymes, or predacious bacterium (Bdellovibrio)

• It is speculated to serve as a permeability barrier to large molecular weight compounds

• May also confer some type of protection against host defense mechanisms to many pathogenic bacteria.

• Helps to maintain the shape and envelope rigidity of some bacterial cells.Can promote cell adhesion to surface.

Periplasmic Space

The periplasmic space refers to a gap that exists between the cell membrane and the outer membrane that is clearly visible in electron micrographs of gram negative cells.  It is rarely seen in GM positive cells and the gap is smaller.

Recent evidence suggest that the PS may be filled with a loose network of peptidogylcan.

The periplasm is a gel like substance that fills the periplasmic space in GM – bacteria. 

In Gram Negative Bacteria

• Contain many proteins that participate in nutrition acquisition.

• Enzymes involved in peptidoglycan synthesis.

• The modification of toxic compounds harmful to the cell.

Capsule

A capsule is a layer of mucoid material that surrounds the bacterial cell, lying outside the cell call.

• When this mucoid layer is thick, well organized, and difficult to remove, it is called a capsule.

• If it is thin, diffuse, and easy to wash off, it is called a slime layer.

• Capsules can easily be seen with a light microscope using a capsule stain.

• Most capsules are composed of a network of polysaccharide fibers that have been secreted from the cell, called the glycocalyx.

•  The amount of capsule produced by a cell depends on the culture conditions.  Growth in high carbon, low nitrogen  medium promote capsule formation.

Function of a Capsule or slime layer:

• Resist desiccation, they contain a great deal of water

• To help the bacterium adhere to surfaces

• prevent or inhibit phagocytosis by a host phagocyte

• Gliding bacteria often produce slime which aids in their motility.