bio 203 lec 3

Why do G+ cells turn purple after Gram staining, but G- cells don't?

- The gram stain process involves the addition of different dyes to differentiate between cell types. - There is a physiological reason for the difference in staining. - Dye called crystal violet is added to the cells. - G+ cells , have a thicker layer around the outside , bind to these crystals very well G- cells which have a lipid layer as there outer most layer , only loosely bind to the crystals - When the iodine gets added next, the losselt bound crystals will release away from the G- cells but remain stuck to G+ cells. Alcohol then washes away these unbound crystals leaving the G+ cells purple and the G- cells clear. Finally,a secondary stain called safranin is added,which binds to all the cells and turns them pink.Because the purple is so much darker,G+ cells will be purple and only the G- cells will look pink under the microscope.

What are some of the functions of the bacterial cell wall? What is the significance of the periplasm?

- the primary function of the cell wall is to provide a protective layer around the cell. - Important to toxic chemicals in the environment and water in the environment - This leads to a feature called turgor for the cell. without the cell wall , this would cause the cell to explode. The cell wall prevents that. - Cell wall defines the shape of the cell. - Cell wall creates a second liquid portion between the cell membrane and cell wall. This is called PERIPLASM ( peri- around; plasm- liquid) - Important for allowing the development of the proton motive(PMF) . The PMF is an electrochemical gradient , which is required for providing energy to the cell.

Describe what you would find in a typical G+ cell wall

A typical G+ cell wall is made up primarily of a thick layer of peptidoglycan, the hallmark molecule of G+ cells. It also includes some teichoic acid (TA) and lipteichoic acid (LTA), which are important for interacting with the environment. Finally, there are some wall-associated proteins, with various functions

Describe what you would find in a typical G- cell wall.

A typical G- cell wall also has peptidoglycan, but only a thin layer, and then outside of that is a second, outer lipid membrane. This second membrane is an asymmetric membrane that contains lipopolysaccharide (LPS), the hallmark molecule of G- cells. There are also some porins and wall-associated proteins.

What is the significance of LPS to infection, and preventing infection, of pathogenic G- bacteria?

In peptidoglycan, NAM and mDAP are both unique molecules, found only in bacteria. In LPS, KDO is unique to bacteria. The significance of these molecules is that they are common molecules that activate an innate immune response; i.e. they are indicative of some kind of general bacterial infection. Furthermore, because the o-antigen is species-specific, it can be used by the adaptive immune system to identify which specific organism is causing an infection. Conversely, lipid A is an endotoxin. This kind of molecule is not designed to be a toxic molecule, but if the cell breaks down, these molecules are harmful to the body and can lead to septic shock.

Describe the general makeup of LPS. What two molecules does a G- LPS always contain?

LPS is made up of three segments, lipid A, the core polysaccharide, and the o-antigen (outer polysaccharide). Lipid A is the part that sticks into the membrane, so it must have a hydrophobic and hydrophilic portion. Therefore, lipid A is made up of 2 NAG sugars, connected to 2 phosphate groups and to at least 5 fatty acids. The core polysaccharide is a black box of sugars, consisting of at least 2 KDO (a unique sugar found only in bacteria). 5 fatty acids and 2 KDO are the minimum requirements of all G- organisms. Above that is the o-antigen, which is a species-specific sugar cluster that gets repeated 30-40 times.

What are the two aminosugars that make up the glycan strands? Where else are these aminosugars found? What is the significance of a b1-4 linkage? What other common molecule utilizes b1-4 linkages?

Peptidoglycan is a molecule made up of long strands of sugars connected by short peptide chains. The long strands of sugars are made up of alternating NAG and NAM. Both of these sugars are derivatives of b-glucose, the type of glucose that makes up cellulose (fiber - the cell wall of plant cells). NAG, or Nacetlyglucosamine, is a b-glucose with an extra side chain on C-2. NAM, or N-acetlymuramic acid, is a NAG with an additional side chain on C-3. NAG is a common sugar, as it is the sugar that makes up chitin, which is the structural sugar that forms the cell wall of fungi and the exoskeleton of arthropods. NAM, on the other hand, is a unique sugar found only in bacteria. This makes it an important molecule in the human body, as its presence is a sign to the immune system of an infection, which therefore leads to an inflammatory response. NAG and NAM are connected by b1-4 linkages; b because they are b sugars, 1-4 because carbon number 1 from one sugar connects to carbon number 4 of the next sugar. These molecules alternate one after another to build a long sugar strand. This b1-4 linkage would be common to all b sugar chains. Therefore, cellulose (aka fiber), which is a long chain of b glucose, uses b1-4 linkages. Chitin, which is a long chain of NAG, uses b1-4 linkages. By contrast, starch, which is a long chain of a glucose, would utilize an a1-4 linkage.

Where and how is peptidoglycan made, and how can this information be used to prevent microbial growth?

Peptidoglycan, which is a component of all bacterial cell walls, must be continuously made as the cell grows and divides. However, to perform chemical reactions necessary to make the cell wall, the cell must have enzymes and energy, which are going to be found inside the cell. Therefore, to build its cell wall, the cell must first make the precursor building block inside the cell, transport it outside, and then add the building block to the existing cell wall. So it starts by making the individual components of the cell wall (NAG, NAM, and the peptide chain), and then links them together (with an extra amino acid tacked on at the end). This building block is then linked to a membrane transporter, which moves it across the membrane. The existing cell wall must then be cut in specific locations, allowing the addition of the new building block. The final step in this process, transpeptidation, is the formation of the crossbridge. This step involves the addition of the extra amino acid onto the peptide chain, which must be broken off to provide the energy to make the crossbridge bond. Because this formation of peptidoglycan involves a common process found in all bacteria, it represents an excellent target for antibiotics. This includes lysozyme, the class of enzymes found in tears, which are unregulated versions of the enzymes that poke holes in the cell wall. Or, it includes a large number of the most common antibiotics, which can block every step of the peptidoglycan synthesis process. This includes b-lactam antibiotics, such as penicillin, which mimic the amino acids at the end of the pentapeptide. When the transpeptidase enzyme binds onto penicillin instead of the pentapeptide, it gets stuck onto the antibiotic and becomes neutralized. If the cell can't form the crossbridges or fill in any of the holes in its cell wall, the cell loses integrity as it fills with water, causing it to explode.

What are the amino acids that are found in peptidoglycan? What is the significance of the D- vs L- isomers?

These individual sugar strands now need to be connected to each other, and this is done by a short peptide chain, made up of 4 amino acids. The 4 amino acids are L-Alanine - D-Glutamic acid - L-Lysine/mDAP - D-Alanine. The L- vs D- isomers represent different structural versions of the amino acids (think of your right vs left hand; while they are made the same way, they are not identical, they are mirror images of each other). The L version is the common version of amino acids found in nature. Bacteria, therefore, must alter two of their amino acids into the D form to build peptidoglycan. The 3rd amino acid is the one that then generates the crossbridge by connecting to the 4th amino acid of another short peptide. If an organism uses mDAP (all G- and ~1/2 the G+ cells), this allows for a direct crossbridge. The organisms that use L-Lys, on the other hand, require an indirect crossbridge.

Cleavage of the L-Ala - D-Glu peptide bond would require what kind of enzyme?

endopeptidase

This endotoxin is responsible for causing septic shock upon its release.

lipid A

What would be the 3rd amino acid residue in a G- bacteria's peptidoglycan?

mDAP