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From CellBiology

Peptidoglycan

Introduction

Peptidoglycan is a polymer peptide found in the exoskeleton of gram positive and gram negative bacterial cells. Peptidoglycan is also known as murein and helps in maintaining the internal osmotic pressure of the cell as well as creating cellular structure and division of the cell wall and cytoskeleton. The mesh-like pattern contains small sized holes called porins where proteins and other molecules can pass in and out of the cell.[1] This layer also contains adhesion sites for other molecules as well as attachment to other bacterial cells. Protein molecules are used during the cell division and growth to insert peptidoglycan chains at specific sites of elongation. The strands of peptides crosslink and contribute in cell wall formation to help in structure, function and protection.[2]

Function

The main function of peptidoglycan is to regulate the cellular shape. Due to the rigid mesh like structure of this layer, it helps to hold together the shape of the cell but also allows the cell to have some flexibility. It also helps in the transport of molecules through its porins and anchored proteins. The anchored proteins can include teichoic acid and Braun’s lipoprotein. These proteins are important in regulation, structure and function to the cell as well.[3]

Osmotic pressure is the pressure acting on the cell. Peptidoglycan maintains the cellular pressure when placed in a hypotonic environment. Without its regulation the cell would take in too much water and burst.[4]

Structure of Peptidoglycan[2]

Structure

The structure of peptidoglycan consists of alternating N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM) molecules. A tetrapeptide is attached to the NAM molecule and contains amino acids. There is also an identical set of peptide bonds cross-bridges. This is essential for the cross linking between the two strands. Depending of the species the peptide bonds and tetrapeptides will differ. Almost all tetrapeptides contain alanine in position one, glutamine at positive two and another alanine on position three although gram positive cells may contain lysine at position three. Peptide bonds will occur between the free amino acid on one chain and the third position alanine on the opposite strand. Gram positive bacteria will use the free amino acid to create a longer cross-bridge. The polymer created by the formation of the peptide bridges can be 12-15 disaccharides in length. A greater peptidoglycan molecule is formed covalently creating a mesh-like pattern in the cell wall.[5]

Synthesis

Peptidoglycan expansion and removal is important in the cell growth and division processes. The production and breakdown of peptidoglycan is constantly occurring throughout the cell. Synthesis of peptidoglycan occurs in 5 stages.

  1. Glucosamine is converted to N-acetyl muramic acid (NAM) which is activated by Uridine triphosphate(UTP) to produce Uridine diphosphate acetyl muramic acid(UDP-NAM).
  2. UDP-NAM pentapeptide is created through enzyme activity.
  3. Bactoprenol is linked to the UDP-NAM pentapeptide through a phosphate link. N-acetyl glucosamine (NAG) is then connected to the pentapeptide creating a disaccharide peptide.
  4. The disaccharide peptide molecule is moved to the outside of the cell by the bactoprenol where it attaches to the previous peptidoglycan chain created.
  5. Still outside the cell, glycan chains create peptide bond transfer with each other which is also known as transpeptidation. This reaction uses no additional energy due to the exchange between the lysine and alanine of the two different strands and the alanine is then released.[6]
Synthesis of Peptidoglycan[3]

Enzymatic Reactions

Enzymatic reactions occur during the processes of creating and destroying the peptidoglycan layer.

Transglycolases

Transglycolases are enzymes that can drive a reaction in forming the cross-bridges between the different NAM and NAG strands. Bactoprenol is used to transport the NAM –NAG molecule across the cell membrane. After it has done its job the Bactoprenol can then be recycled and reused for the next cross bridging process. If Bacitracin is present in the cell than the Bactoprenol will not be recycled, causing the cell to not be able to repair the peptidoglycan layer. Lysozyme is an autolysin that helps in hydrolyzing the bond between the NAM and NAG molecules and is also an important molecule in the degradation of the cell wall.[7]

Transpeptidases and Carboxypeptidases

Transpeptidases and carboxypeptidases are proteins used to catalyze the cross linking reaction of the peptidoglycan. These proteins are bound to the cell membrane and eliminate alanine molecules that have not reacted during the process. These proteins are also known as Penicillin- Binding Proteins (PBPs) because antibiotics are able to attach to them (see Antibiotics). If the PBPs are inhibited than there will be irregularities in the cell wall leading to cellular death. PBPs are used for the extension of peptidoglycan, the curving and shape of the cell wall and to create the starting septum for cellular division. MreB, MreC, FtsZ and FtsI all play a role in regulating and inserting peptidoglycan synthesis during cellular division. These proteins are used to insert peptidoglycan in the pre- existing cell as well as helping in the synthesis of peptidoglycan in the new cell. [8]

Degradation

Antibiotics

Antibiotics are used in destruction of the cell wall. They are used to target different aspect of the peptidoglycan layer by affecting it through proteins, enzymes and molecules that help in its synthesis. Some antibiotics are produced to work directly on the peptidoglycan layer to destroy the cell. Penicillin is an antibiotic that attaches to the PBPs and destroys the synthesis of the peptidoglycan, leading to cell death. Vancomycin and cytoserine are used in destroying the cross-bridges of the peptidoglycan which breaks it down and destroys the cell. Bacitracin works on prohibiting the movement of the precursor molecules like Bactoprenol; this will also stop the synthesis of peptidoglycan.[9]

Autolysins

Autolysins are enzymes that can help in creating and destroying the peptidoglycan layer of bacteria. They work by breaking down the peptide bonds between NAM and NAG molecules which breaks down small portions of the peptidoglycan layer so that the cell can divide and grow. Teichoic acid is used in gram positive cells to regulate the production of autolysins. If the teichoic acid production is inhibited the autolysin will continue breaking down the peptidoglycan layer causing the cell to burst due to increased osmotic pressure.[10]

Cell Starvation

Cell starvation can occur if the cell is put in an atmosphere that lacks enough nutrients. When this occurs the new production of peptidoglycan will cease and the wall becomes less rigid. Overtime, the cell wall will break down and cause the cell to burst.[11]

Gram Staining

The actual gram staining technique was created by a man named Hans Christian Gram during the 1980s. He founded this staining process while studying patients who had died from bacterial pneumonia. He cultured their lung tissue to find more information about the bacterial cells. After studying bacterial cells he understood that they could be divided into two groups by a staining process which helped in classifying them. Based on cell wall structures like peptidoglycan he created the gram positive and gram negative groups.[12]


Gram positive and gram negative cells in muscle tissue[4]

Gram Positive Bacteria

Gram positive bacteria will form a stronger peptidoglycan wall by creating a three dimensional mesh work with multiple layers. There walls tend to be thicker, stronger and more rigid. Gram positive bacteria also contain teichoic acid which is covalently linked to the peptidoglycan layer and lipoteichoic acids linked to the cell membrane. Teichoic acids are water soluble and help in maintaining cellular viability through autolysin regulation while lipoteichoic acids are fatty acids acting as antigens. Both act as virulence factors for the cell. Gram positive cells retain a purple color due to the thick peptidoglycan layer, teichoic and lipoteichoic acids.[13]

Gram Negative Bacteria

Gram negative bacteria form a single layer of peptidoglycan wish shorter cross links. This creates a less rigid layer and is much thinner. These types of bacteria contain lipopolysaccarides (LPS) which exclude hydrophobic molecules and protects the cell. Hydrophilic molecules can pass in and out of the cell due to the porins in the cell wall. Braun’s lipoprotein is an important molecule in gram negative bacteria. The gram negative cells will turn a red color after gram staining due to the thin peptidoglycan layer and LPS.[14]

Gram Staining Procedure

  1. Heat fix a bacteria sample to a microscopy slide
  2. Add Crystal violet to the slide for about a minute period
  3. Add Iodine to the slide for 30 seconds
  4. Rinse slide lightly with water
  5. Add a decolorizer like Ethanol to the sample. During this step, gram negative bacteria will lose the purple color from the crystal violet but the gram positive bacteria will retain the purple color.
  6. Add Safranin to the bacteria sample for another thirty seconds
  7. Rinse with water and lightly pat dry without smudging. The gram negative cells will then turn a red color.[15]

Glossary

  • Alanine- one of the crystalline non-essential amino acids formed by the hydrolysis of proteins
  • Antibiotics- substance produced to inhibit or kill another microorganism
  • Braun’s lipoprotein- the most abbundant membrane immunilogical protein found in gram negative cells
  • Crystal violet- triphenylmethane dye found in gentian violet
  • Elongation- step during DNA synthesis where the chain goes through elongation
  • Ethanol- toxic, volatile liquid used often used as a solvent and decolorizer
  • Exoskeleton- supportive external covering
  • Glutamine- crystalline amino acid found in plants and animals producing glutamic acid
  • Hydrophilic- object having a strong affinity to water
  • Hydrophobic- object having a lack of affinity to water
  • Hypotonic- object having a lower osmotic pressure than the liquid or medium surround it
  • Lipopolysaccarides- large molecule consisting of chemically bound sugars and lipids
  • Lipoteichoic acid- cell surface adhesion used in fighting infection in gram positive cells
  • Lysine- crystalline essential amino acid synthesized by protein hydrolysis
  • N-acetylglucosamine (NAG)- monosaccharide derivative of glucose used to chemically form the peptidoglycan layer
  • N- acetylmuramic acid (NAM)- ether molecule derivative from NAG used to chemically form the peptidoglycan layer
  • Osmotic pressure- the maximum pressure that develops in a solution separated from a solvent by a membrane permeable only to the solvent
  • Peptide- molecule produced by the linking of an amino group of an amino acid to the carboxyl group of another amino acid
  • Porin- protein carries that limits the free diffusion of molecules through the membrane
  • Safranin- red synthetic dye used in biological staining
  • Teichoic acid- strongly acid polymers containing glycerol found in cell walls and membranes of gram positive cells
  • Virulence factor- the ability of an organism to cause disease which can be based on adhesion, toxins and invasion

Information taken from Medline Plus Medical Dictionary.[16]

References

  1. A. Rose, R. Poole, J. F. Wilkinson. Advances in Microbial Physiology. Elsevier, 1995, pg. 23-81.
  2. P. Murray, K. Rosenthal, M. Pfaller. Medical Microbiology. Elsevier Health Sciences, 2008, pg. 10-18.
  3. A. Rose, R. Poole, J. F. Wilkinson. Advances in Microbial Physiology. Elsevier, 1995, pg. 23-81.
  4. P. Engelkirk and G. R. Burton. Burton's Microbiology for the Health Sciences. Lippincott Williams & Wilkins, Edition 8, 2006, pg. 58-60.
  5. A. Rose, R. Poole, J. F. Wilkinson. Advances in Microbial Physiology. Elsevier, 1995, pg. 23-81.
  6. P. Murray, K. Rosenthal, M. Pfaller. Medical Microbiology. Elsevier Health Sciences, 2008, pg. 10-18.
  7. P. Murray, K. Rosenthal, M. Pfaller. Medical Microbiology. Elsevier Health Sciences, 2008, pg. 10-18.
  8. P. Engelkirk and G. R. Burton. Burton's Microbiology for the Health Sciences. Lippincott Williams & Wilkins, Edition 8, 2006, pg. 58-60.
  9. Harwood, Collin. Bacillus. Springer, Edition 2, 1989, pg. 238-241.
  10. P. Murray, K. Rosenthal, M. Pfaller. Medical Microbiology. Elsevier Health Sciences, 2008, pg. 10-18.
  11. P. Engelkirk and G. R. Burton. Burton's Microbiology for the Health Sciences. Lippincott Williams & Wilkins, Edition 8, 2006, pg. 58-60.
  12. R. Colwell and R. Grigorova .Methods in Microbiology. Academic Press, Vol. 19, 1978, pg.5-9.
  13. Harwood, Collin. Bacillus. Springer, Edition 2, 1989, pg. 238-241.
  14. Harwood, Collin. Bacillus. Springer, Edition 2, 1989, pg. 238-241.
  15. R. Colwell and R. Grigorova .Methods in Microbiology. Academic Press, Vol. 19, 1978, pg.5-9.
  16. Medline Dictionary[1]

Text Books

  • A. Rose, R. Poole, J. F. Wilkinson. Advances in Microbial Physiology. Elsevier, 1995, pg. 23-81.
  • Harwood, Collin. Bacillus. Springer, Edition 2, 1989, pg. 238-241.
  • P. Engelkirk and G. R. Burton. Burton's Microbiology for the Health Sciences. Lippincott Williams & Wilkins, Edition 8, 2006, pg. 58-60.
  • P. Murray, K. Rosenthal, M. Pfaller. Medical Microbiology. Elsevier Health Sciences, 2008, pg. 10-18.
  • R. Colwell and R. Grigorova .Methods in Microbiology. Academic Press, Vol. 19, 1978, pg.5-9.

Dictionary

  • Medline Dictionary [5]

PUBMED

  • M. Green and P. Karp. "The outcomes of pathway database computations depend on pathway ontology". Nucleic Acids Res. 2006, Volume 34,3687–3697. [6]
  • E. Schröpfer,S. Rauthe, and T. Meyer. "Diagnosis and misdiagnosis of necrotizing soft tissue infections: three case reports"Cases J. 2008,Volume 1, 252. [7]