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Cell Wall (cont.)

©2001 Timothy Paustian, University of Wisconsin-Madison

The Gram Negative Cell Wall

G^- cell walls have a more complicated structure. There are two separate areas with an additional membrane besides the cellular membrane. Outside of the cytoplasmic membrane (CM) is a open area called the periplasmic space. Beyond this is a thin layer of peptidoglycan. Finally, external to the peptidoglycan is an additional membrane, the outer membrane (OM).

Figure 1 - Gram negative cell wall

Peptidoglycan

The peptidoglycan in G^- cells contains less cross-linking with no peptide interbridge. Covalently bound to the peptidoglycan is Braun's lipoprotein, which has a hydrophoic anchor in the OM. This strongly binds the peptioglycan to the outer membrane.

Outer Membrane

The OM has been extensively studied due to its large role in the virulence (ability to cause disease) of G^- bacteria. The OM is a Lipid bilayer similar to the cell membrane, containing lipids and proteins, but also lipopolysaccharides. The membrane has distinctive sides, with the side that faces the outside containing all the lipopolysaccharide.

Lipopolysaccharide (LPS)

LPS is composed of two parts, Lipid A and the polysaccharide chain that reaches out into the environment. Lipid A is a derivative of 2 NAG units with up to 7 fatty acids connected to it that anchor the LPS in the membrane. Attached to Lipid A is a conserved core polysaccharide that contains, KDO, heptose, glucose and glucosamine sugars. The rest of the polysaccharide consists of repeating sugar units and this is called the O-antigen. The O-antigen gets its name from the fact that it is exposed to the outer environment and host defenses will often raise antibodies to this structure. The O-antigen varies between species and even between various isolates of a species. Bacteria protect themselves against the hosts defenses by varying the make-up of the O-antigen.

Figure 2 - Lipopolysaccharide

LPS confers a negative charge and also repels hydrophobic molecules. Some G^- species live in the gut of mammals and LPS will repel fat soluabilizing molecules such as bile that the gal bladder secretes. The O-antigen is also involved in recognition by certain bacteriophage (viruses that infect bacteria).

LPS is medically important because it has activities in humans. Free LPS in solution is toxic and is called endotoxin. The compound, when released from bacterial cells is toxic to mammals creating a wide spectrum of physiological reactions such as:

• The induction of a fever. Endotoxins are said to be pyrogenic.

• Changes in white blood cell counts

• Leaking blood vessels

• tumor necrosis

• Dropping blood pressure leading to vascular collapse and eventually shock

• At high enough concentrations endotoxin is lethal

Outer membrane proteins

There are fewer different types of outer membrane proteins when compared to the cytoplasmic membrane, but they are in higher abundance. I will only focus on the porins because of their importance in OM permiability. Porins are proteins that form pores in the outer membrane wide enough to allow passage of most small hydrophilic molecules. This allows migration of these moleules into the periplasmic space for transport across the cytoplasmic membrane. Larger or hydrophobic molecules cannot penetrate the outer membrane.

Functions of the outer membrane

• Confers negative charge to cell

• Selective barrier, pores for entrance of hydrophilic molecules

• Phage receptors - although this is a function the cell would rather not have.

• Pathogenic properties

• Stabilizes mating cells

• Keeps Enzymes in periplasm

Periplasm

This is the space inbetween the peptidoglycan and the outer membrane which contains many different proteins. These proteins fucntion to detect the environment and transport needed nutrients into the cell. Some examples of periplasmic enzymes include..

1. hydrolytic enzymatic enzymes
   • phosphatases - degrade phosphate containing compounds
   • proteases - degrade proteins and peptides
   • endonucleases - degrade nucleic acids
2. binding proteins - recognize specific solutes and transport across membrane:
   • sugars
   • amino acids
   • inorganic ions
   • vitamins

3. chemoreceptors - helps cell interpret chemical composition of its environment

4. detoxifying enzymes - alter harmful agents before they get into cell, example beta-lactamase

5. osmotic protection. - When the cell is put in high osmolarity (high solute concentration) it causes water to flow out of the cell. To protect themselve, bacteria synthesize small molecules to balance the osmotic stress. These are called compatible solutes and they accumilate in the periplasm.

The table summarizes the difference between G- and G+ cell walls

Figure 3 - A comparison of cell wall types

Bacterial Lacking Cell Walls

For most bacterial cells, the cell wall is critical to cell survival, yet there are some bacterial cells which do not have cell walls. Mycoplasma species are one example and they are very wide spread. They are obligate intracellular pathogens (they can only survive inside of their host). Some of this dependency is based on the lack of cell walls. As an example, they are quickly killed if placed in an environment with very high or very low salt concentrations. Due to the lack of a cell wall, Mycoplasma have unusually tough membranes that are more resistant to rupture than other bacteria. The presence of sterols in the membrane contribute to their durability. Mycoplasma are also pleomorphic

Some bacteria may mutate or change because of extreme nutritional conditons to form a cell wall-less form or L-forms. This phenomenon is observed in both G⁺ and G^- species. These forms may result from partial or complete loss of the cell wall. L-forms have a varied shape and are sensitive to osmotic shock

Function of the cell wall

• The cell wall confers a shape to the bacteria. If you remove it, the cell will become an amorphous blob. Also, certain bacteria have long appendages that increase the surface area of the cell. This allows the cell to live in very dilute environments, yet still scavange what it needs.

• The cell wall is also directly in contact with the environment. Its interaction with the outside world may determine the successful survival of the cell. Two examples to think about are....

  • Interaction with a host cell in the intestine to begin attachment

  • The binding of a virus that infects the bacterial cell.

• The cell wall is also involved in many pathogenic properties of the bacteria. In both G⁺ and G^- species, the cell wall is very important in attachment to specific host cells during the infection stage. The cell wall can even be a pathogenic determinate.

• The cell wall can act as a barrier to some molecules. G⁺ cell walls have a negative charge and are hydrophilic due to the presence of teichoic acid. This acts as a barrier to molecules with a positive charge. G^- cells walls are very hydrophilic due to the presence of LPS in their outler membrane. The LPS acts as a barrier to hydrophobic molecules and is why these organisms are resistant to hydrophobic compounds like Crystal Violet and bile.

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