-
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
-
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)
-
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
-
peptidoglycan sacculus
- one large macromolecule
- glycan chains run around circumference of the cell
- peptide crosslinks connect adjacent glycan strands
-
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
-
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
-
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
-
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
-
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
-
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
-
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
-
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
-
β-lactams resistance
mediated by β-lactamase enzymes - break the ring and inactivate the antibiotic
-
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
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