April 18, 2008

Cell Walls in Microorganisms

Posted in Microbiology tagged , , , , at 10:28 pm by D. Borst

Gram Positive Stain of S. AureusLipid bilayers are relatively weak. Certainly there are stronger forms of these membranes, but as implied by the fluid mosaic model these membranes sacrifice rigidity and strength for the ability to allow proteins to freely disperse within the membrane. A consequence of this is that differences in pressure on the two different sides of the membrane can quickly lead to membrane rupture.

This presents a problem for life, because in order to complete the processes needed many proteins, ions and other molecules are required in a very small space. However, to have all these molecules concentrated within the cell would cause there to be a high osmotic pressure for water to flow into the cell. If the cell is not able to control this water absorption, it will swell to the point where it bursts. In animals, the solution is to keep the cells immersed in a solution that has the same number of osmolites dissolved in it as the cells do. Thus the cell cytoplasm is isoosmotic with the extra-cellular environment.

Not all organisms solve the problem in this manner however. Some microorganisms actively excrete water to keep their size down. Plants create rigid cell walls around their cells. This cell wall supports the lipid bilayer and allows it to resist the pressures that are acting upon it. The greater pressures within the cell then will not rupture the cell membrane.

The same tactic is taken by many forms of microorganisms. The cell walls that these organisms create allow them to withstand considerable turgor pressure, up to 2 atm in E. coli. These microorganisms often do not need to be able to deform their cellular shape, so having a cell wall does not need to be a liability as it would be in animals. Furthermore, the presence of a cell wall provides other advantages, in that it provides a barrier to viruses and other bacteria that may try to harm the organism. In the rest of this post, I will be going into some of the basics of cell wall morphology in both Bacteria and Archea.

For starters, bacteria are often classified in two different groups, as either being Gram Positive or Gram Negative. This designation has to do with a bacterium’s reaction to a differential staining technique called the Gram Stain. The gram stain first uses Crystal Violet, which stains all cells with a cell wall a violet color. However, then the cells are exposed to ethanol or acetone. If the ethanol or acetone can access the crystal violet in the cell walls, then it will deactivate the crystal violet and the cell will become colorless. However, in gram positive cells, the properties of the cell wall do not allow ethanol or acetone to pass, and thus the violet stain is retained. In Gram negative cells the ethanol or acetone passes through and the color is lost. The gram negative cells are then resolved using a secondary stain, safranin. Gram positive cells will retain the primary stain, Crystal Violet, and will not take up the safranin. They will remain a dark violet color after the process. Gram negative cells will have lost their Crystal Violet stain, and will take up the safranin, and will appear reddish.

The stain is particularly useful because there are few species of Bacteria that fall into neither category. The gram stain provides a test that is quick and cheap, as well as dividing the field of possible microbes by a factor of about two. However, it is important to remember that while exceptions are rare, they do exist. There are gram negative bacteria that are such for other reasons than the morphology of their cell wall: One example is Mycobacterium tuberculosis which has a cell wall that resembles most gram positive bacteria, but contains many lipids which does not allow it to take up the stain.

Gram Positive Cells

Gram positive cells generally have a thick cell wall surrounding the cell membrane. This cell wall is formed of peptidoglycan, a polymer of L and D peptides along with the sugars N-acetylglucosamine (NAG) and N-acetylmuric acid (NAM). A simple schematic of the gross morphology of an idealized gram positive cell is shown below:

Gram Positive

The structure of the peptidoglycan cell wall is complex and varies with different strains of bacteria. Basically there will be a glycan backbone that is a polymer of NAG and NAM molecules, and the NAM’s will have a tetrapeptide peptide chain hanging off of them. In gram positive bacteria, the peptide chains are connected by way of a peptide interbridge. Together, the complex forms a large lattice like sheet that surrounds the cell. This is represented in the above images, red representing NAM sugars, Orange NAM sugars, and the purple circles the complex of proteins. The strength and rigidity of the cell wall varies between species both by way of the nature of the peptide interbridge and peptide sequence that is attached to the glycan backbone, and by how many interbridges are actually present. In the above image, the lattice structure is very regular, and saturated, however this need not be the case.

As mentioned above, peptidoglycan contains by the L and D entantiomers of amino acids. This indicates that the protein strands are constructed in a different manner than others in the cell, as D amino acids cannot be handled by the ribosome. Furthermore, there is great diversity in the exact nature of the peptidoglycan linkages in gram positive bacteria. he glycan backbone is completely conserved among diferent species, and there is little variation in the tetrapeptide sequence. Most of the variaiton that exists is in the sequence of the interbridge. In all, there are more than 100 different peotidoglycan types known.

The shape and size of the bacterial cell is thought to be determined by the details of the peptidoglycan crosslinking and the length of peptidoglycan strands. T
There are two types of Bacteria

While the cell walls consists mainly of peptidoglycan, there are other constituents present in varying amounts among different species. The second major constituent (though it is still present is much lower quantities than peptidoglycan) are teichoic acids. Teichoic acids are embedded in the cell wall much like proteins are embedded in the cell membrane. They have a number of functions including to capture calcium and magnesium ions which can then be transported into the cell.

As mentioned above, Gram Positive cells remain purple in the presence of ethanol. This is thought to happen because the Crystal Violet complex forms inside the cell wall. When exposed to ethanol, the thick cell wall is dehydrated, and the pores in the wall shrink. The insoluble Crystal Violet is then incapable of being extracted, and the cell remains purple.

Gram Negative Cells

Gram negative bacteria display a radically different morphology than Gram positive cells. Gram negative cells have both an inner and an outer membrane like the mitochondria. The space between the two membranes is known as the periplasmic space, and it is there that a thin layer of peptiodoglycan exists. Below is a simple schematic of the gross morphology of an idealized gram-negative cell.

Gram Negative

The structure of the peptidoglycan in the gram negative cell is slightly different than the structure in the gram positive cell. The most obvious difference is that there is simply less peptidoglycan in the gram negative cell. This has the effect of allowing alcohol in the gram stain to leach out the crystal violet, which is why these cells loose their color when so exposed. There are also some differences in the structure of the peptidoglycan in gram negative cells. The peptidoglycan strands in gram negative bacteria do not use interbridges to connect the tetrapeptides on different strands. Rather, the tetrapeptide strands are directly linked to one another.

The outer membrane of gram negative cells has large porin proteins in it to allow molecules to apprach the inner membrane. This allows useful substances from outside the cell to enter the cell. Furthermore, the outer membrane does not consist solely of lipids. Many of the lipids are ligated to polysaccharide chains, and this amorphous fourth layer is often referred to as the lipid polysaccharide layer.

The polysaccharides in the LPS consist of three portions, a lipid (reffered to as lipid A) connected by amine ester linkage to NAG-Phosphate, which then connects to the Core Polysaccharide. The core polysaccharide then connects to the O-specific Polysaccharide. All of these components vary among different species, but this general order is maintained.

Notably, on the outer leaflet of the outer membrane of such gram negative bacteria, there are no phospholipids. Rather, the glycan groups take the place of the phosphate. On the inner leaflet there are phospholipids however. Note that this is very different from most lipid bilayers, which generally have phospholipids on either side.

The LPS layer can also be toxic to other organisms. Endotoxin is one case of this, where it is the lipid portion of a unit of the LPS that creates the toxic reaction.

Cell Walls in Archea

Peptidoglycan only exists in the Bacteria branch of the tree of life. However, as in Bacteria almost all Archea contain some sort of cell wall. Some species of Archea have cell walls, but they use a different compound, called pseudopeptidoglycan. Other species in Archea use complexes of other polysaccharides, glycoproteins and proteins. The S-layer is the most common type of cell wall in Archea however.

The S-layer is a paracystalline layer with generally hexagonal symmetry. It is generally composed of a identical proteins or glycoproteins, which vary between species. The nature of the molecule that composes the S-layer allows it to self assemble, however it is poorly conserved and varies widely between species.

Cells without cell walls.

A few species of bacteria exist that do not have cell walls. These must beat the osmotic game in a different way if they are to persist. There are several strategies that they use to overcome their disadvantages. Some simply create incredibly strong and rigid membranes, reinforced with sterols to make them stiff. This has basically the same effect as a cell wall, however it provides some advantages in allowing the cell more ready access to the nutrients in the extra-cellular space.

Others simply live in environments that are iso-osmotic. Some organisms have evolved to be isoosmotic with the interstitial space of animals, allowing them to live sans wall outside. Many of these have a cell wall when they are out of the body, that is destroyed by lysozyme when they enter.

The information for this post was attained during Dr. Popa’s April 8th Lecture at PSU, from Brock’s Biology of Microorganisms and from several wikipedia posts, including the article on Gram staining, and the S-Layer and from AJ Cann’s post on the Gram Stain on MicrobiologyBytes. The images in this post were created by me and are hosted at bayimg.com. If you so desire, you are free to use them and modify them in any way that you desire.

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1 Comment »

  1. Jaxon said,

    awesome. great for prokaryote morphology introduction/revision. good also for identification. good work on the creative commons diagram too.


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