Peptidoglycan

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Peptidoglycan

• Reading requirement Chapter 4

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Gram stain

• An important differential staining procedure
  widely used in bacteriology
• On the basis of their reaction to Gram stain
  bacteria can be divided into two major groups
  Gram-positive and Gram-negative
• The differences in reaction to Gram stain is due
  to differences in the cell wall structure of
  gram-positive and gram-negative cells,
  particularly, the composition of peptidoglycan
  (PG)
• In Gram-positive bacteria, as much as 90 of the
  cell wall consist of peptidoglycan layers (up to
  about 30 layers). In Gram-negative bacteria, only
  about 10 of cell wall is peptidoglycan (1 layer)

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Peptidoglycan (PG, also called Murein)

• Chemically unique, rigid structural component of
  cell wall found in bacteria
• Largest molecule in bacterial cell and constitute
  50-90 of Gram-positive cell wall and lt10 of
  Gram-negative cell wall dry weight
• Strengthening element, provide shape of cells and
  preserve integrity of the cytoplasmic membrane
  from rupture in medium or low osmolarity
• Contains compounds unique to the microbial world
  (D-amino acids, diaminopimelic acid (DAP),
  N-acetyl muramic acid)

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Types of peptide subunits in peptidoglycan

• Type A -L-Ala-D-Glu-DAP-D-Ala-
• All Gram-negative bacteria, a few gram-positive
  bacteriaBacillus megaterium, Corynebacterium
  diphtheriae
• Type A modified -L-Ala-D-Glu (NH2)-DAP-D-Ala-,
  Bacillus subtilis (Gram)
• Type B -L-Ala-D-Glu(NH2)-L-Lys-D-Ala-
• Staphylococcus aureus
• Streptococcus pyogenes
• Streptococcus faecalis
• Lactobacillus casei
• Lactobacillus acidophilus

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Type of peptide subunits in PG

• Type C -L-Ala-Gly-L-Lys-D-Ala
• Micrococcus lysodeikticus
• Sarcina lutea
• Type D-L-Ala(Gly)-D-Glu-L-homoserine-D-Ala
• Corynebacterium poinsettiae
• Type E -L-Ser-D-Glu-L-Ornithine-D-Ala
• Butyribacterium rettgeri
• Note bacteria with B, C, D, and E peptide
  subunits are Gram-positive

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Types of cross linking

• Type I direct bond between D-Ala and DAP
• E. coli and all other Gram-negative bacteria

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Types of Cross linking

• Type II bridge between D-Ala and Lysine.
• Staphylococcus aureus
• Other types of bridge found
• -L-Ala-L-Ala-
• Streptococcus pyogenes
• -L-Ala-L-Ala-L-Ala
• Micrococcus roseus
• -Gly-Gly-L-Ser-Gly-Gly-
• -Gly-L-Ser-Gly-Gly-Gly-
• L-Ser-Gly-Gly-Gly-Gly-
• Staphylococcus epidermidis

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Types of cross linking

• Type III head to tail assembly of several
  peptides, each with sequence of amino acids the
  same as the peptide subunit
• The tetrapeptide is cleaved form the N-acetyl
  muramic acid by an enzyme -N-acetyl-muramyl-L-ala
  nine amidase
• Example Micrococcus lysodeikticus, Sarcine lutea

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Types of cross linking

• Type IV linkage between second amino acid and
  the 4th amino acid
• Example Corynebacterium poinsettiae

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Cell Division and PG Biosynthesis
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Enzymes active on peptidoglycan

• Transglycosylases
• N-acetylmuramidase breaks the bound between
  N-acetylglucosamine and N-acetylmuramic acid at
  the reducing end of N-acetylmuramic acid to
  permit insertion of newly synthesized
  disaccharide pentpeptide units into the growing
  glycan strand
• Lysozyme an N-acetylmuramidase present in
  animals (prominent in tears and egg white) active
  against invading bacteria by attacking the
  peptidoglycan of the bacteria
• N-acetylglycosaminidase breaks the bond between
  N-acetylglycosamine and N-acetylmuramic acid at
  the reducing end of N-acetylglucosamine

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Enzymes active on PG
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Enzymes active on PG

• Transpetidase cleaves the bond between
  D-Ala-D-Ala and forms a new bond between the
  carboxyl groups of the remaining D-Ala (the 4th
  amino acid in the peptide) and the ? amino acid
  group of DAP. The energy released by the cleaving
  bond between D-Ala-D-Ala is used to form the new
  bond. Cross linking is a more complex process for
  Gram-positive bacteria because bridges are
  inserted between the glycan strands.

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Action of Transpeptidase
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Enzymes active on PG

• Carboxypeptidase cleaves the terminal (5th
  D-Ala) from the peptide subunit but does not form
  a bond between the remaining D-Ala and the 3rd
  amino acid of the peptide subunit of the
  N-acetylmuramic acid of another glycan strand and
  thus functions in regulating the degree of
  cross-linking of glycan strands.

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Enzymes active on PG

• Endopeptidase breaks the bond that cross-links
  the glycan strands (reverse action of
  transpeptidase) to create sites into which newly
  synthesized peptidoglycan units can be inserted
  in the growing or dividing cell. It functions in
  concert with transglycoslases. e.g., Lysostaphin.
  

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Enzymes active on PG

• N-acetylmuramyl L-alanine amidase breaks the
  bond between N-acetylmuramic acid and the 1st
  amino acid (L-Ala) of the peptide subunit to
  yield free tetrapeptide subunits. These may be
  used for producing the bridges of the type 3
  peptidoglycan bridge.
• Autolytic enzymes (autolysins) causes breakdown
  of peptidoglycan leading to cell lysis (cell
  death) when enzymes are unregulated or
  uncontrolled. Enzymes that may act as autolysins
  include muramidases, glucosaminidases,
  endopeptidases and amidases.

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Bacterial cell shape

• The ratio between transpeptidation (by
  transpeptidase) and carboxypeptidation (catalyzed
  by carboxypeptidase) is important in generating
  degrees of cross-linking and a three-dimensional
  structure of the cell wall (angles, curves,
  etc.), which determines the shape of bacterial
  cells.

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Gram-staining

• In the Gram stain, an insoluble crystal
  violet-iodine complex is formed inside the cell,
  and this complex is extracted by alcohol from
  gram-negative but not from gram-positive
  bacteria. Gram-positive bacteria, which have very
  thick cell walls consisting of several layers of
  peptidoglycan, become dehydrated by the alcohol.
  This causes the pores in the walls to close,
  preventing the insoluble crystal violet-iodine
  complex from escaping. In gram-negative bacteria,
  alcohol readily penetrates the lipid-rich outer
  layer, and the thin peptidoglycan layer also does
  not prevent solvent passage thus the crystal
  violet-iodine complex is easily removed.

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PG of Gram-Negative Bacteria
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PG of Gram-Positive Bacteria
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Cell Envelope