7. Bacterial Cell Walls

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7. Bacterial Cell Walls
2011-10-04 14:11:20
PMB 112 midterm1

general microbiology midterm 1
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  1. gram positive
    • thick layer of peptidoglycan
    • teichoic acids - sugar/amino polymers that give overall negative charge to the cell surface and bind Ca++ and Mg++ for eventual uptake, ~50% of mass of cell envelope
    • lipoteichoic acid - covalently anchored in cytoplasmic membrane
  2. gram negative
    • thin layer of peptidoglycan
    • outer membrane attached to PG via lipoproteins, has porins (size dependent solute exclusion) for permeability
    • outer leaflet has lipopolysaccharide (LPS)
  3. structure of LPS
    • lipid A: fatty acids linked to glucosamine phosphate via ester amine bond, called endotoxin
    • core polysaccharide: contains hexoses, heptoses and ketodeoxyoctonate
    • O-specific polysaccharide (O-antigen): species-specific, made of hexoses in branched units that repeat

    protects cells from bile salts, hydrophobic antibiotics and complement activation
  4. peptidoglycan sacculus
    • one large macromolecule
    • glycan chains run around circumference of the cell
    • peptide crosslinks connect adjacent glycan strands
  5. repeating unit of peptidoglycan
    • NAG+NAM in β(1,4) linked chains
    • Glycan chains attached to each other by peptide crosslinks
    • contains D-amino acids
    • diaminopimelic acid found only in Bacteria - has two amino groups allowing peptide crosslinking
  6. peptide crosslinks
    • different in different species
    • gram - :between DAP and D-Ala
    • gram +: between L-Lys and D-Ala or short peptide interbridge forms crosslink
    • more crosslinking yields greater rigidity
  7. bacterprenol
    • hydrophobic carrier holds PG subunits in the inner membrane until they are added to the exisiting PG scaffold
    • moves PG subunits across the cytoplasmic membrane
  8. peptidoglycan synthesis
    • 1) NAM pentapeptide attached to bactoprenol to make lipid I
    • - synthesized in cytoplasm and inner membrane by Mur pathway
    • 2) NAG attached to lipid I to form lipid II
    • - done by MurG enzyme in cytoplasmic membrane
    • 3) Lipid II translocated to periplasmic side of cytoplasmic membrane, carried by bacterprenol, mediated by flipase enzymes
    • * next two steps must occur outside the cell wall without the presence of ATP
    • 4) transglycosylation = linking of NAG of new monomer onto NAM of existing chain by PBP1
    • - bacterprenol returns to original orientation to be recycled
    • 5) transpeptidation = crosslinking new PG strand into a sheet via its peptide side chain into a PG sheet by PBP1, PBP2 and PBP3
  9. Penicillin-binding proteins
    • located in cytoplasmic membrane with soluble portion facing out or entirely in periplasmic space
    • high-MW PBPs (1,2,3) - perform transglycosylation or transpeptidation outside the cytoplasmic membrane
    • low-MW PBPs - peptidases that remove amino acids or break the peptide crosslinks in mature PG
    • lytic transcylcosylases - break the β(1,4) linkages between NAG and NAM
    • functional redundancy = several enzymes perform each reaction
  10. PG osmotic lysis experiments
    • if cells are in hypotonic medium when PG is disrupted they will lyse due to osmotic pressure
    • if cells are in isotonic medium when PG is disrupted, the IM stays intact and protoplasts are formed
    • PG sacculus retains its shape after cell lysis, but PG is not a static structure - protects against lysis
  11. penicillin
    • In normal transpeptidation:
    • serine residue on PBP forms covalent intermediate with a pentapeptide, released free D-ala
    • the PBP D-ala intermediate is attacked by the NH2 group of DAP on a neighboring peptide to form a crosslink and release the PBP enzyme

    • blocks the transpeptidation step of PG synthesis:
    • mimics the D-ala-D-ala residues of PG peptides and is attacked as a substrate
    • β-lactam ring is broken during attack by the PBP, forming an irreversible intermediate with the PBP
  12. How do β-lactams kill bacteria?
    • PG is continuously synthesized and broken down
    • the the presence of a β-lactam, transglycosylation proceeds normally but the glycan strands are not crosslinked, so the PG becomes weaker
    • PG lysis proceeds normally, balance is shifted toward breakdown of the cell wall
  13. β-lactams resistance
    mediated by β-lactamase enzymes - break the ring and inactivate the antibiotic
  14. How do bacteria maintain shapes during growth and division?
    • PBPs help to generate characteristic cell shapes
    • by knocking out one or several PBP genes at a time, investigators have found several effects on cell shape
    • PBP localization conincides with predicted functions