Flashcards in Lecture 3 Deck (31):
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Gram Stain
Differential staining procedure
2 Groups: G+ and G-
Difference to Gram stain is due to cell wall composition of Peptidoglycan (PG)
90% of cell wall is PG for G+ (up to 30 layers)
10% cell wall is PG for G-
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Steps to Gram staining
1. Flood heat-fixed smear with crystal violet
All cells purple
2. Add iodine solution - All still purple
3. Decolourize with alcohol - G- lose their colour, G+ still purple
4. Counterstain with safranin - G+ are purple, G- are pink/red
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Membrane of G+ and G- Bacteria
G+ have PG and cytoplasmic membrane
G- are like G+ except they have a outer membrane made of Lipopolysaccharide and protein
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Peptidoglycan (PG, Murein)
unique chemical, structural component of bacteria cell wall
Largest molecule, 50-90% of G+ and , <10% of G- dry weight
Provides strength and shape to cells
Contains unique D-AA, diaminopimelic acid (DAP), and N-acetyl muramic acid
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PG and osmolarity
PG makes cell wall rigid
Protects bacteria from external osmolarity changes
Digest PG with lysozyme removes protection and cells dies from osmolarity change
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Sacculus
PG sack retains shape after bacteria is disturbed
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Structure of PG
subunits are sugar (glycan) and peptides
Wall Glycan: N-acetyl muramic acid or glucosamine (NAM, NAG)
Wall Peptide: L-Alanine, D-isoglutamate (G-) or D-isoglutamine (G+), Lysine or meso-DAP, 2 D-Alanine
Free carboxyl on glutamate is ofter amidated in G+
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Wall Glycan
NAG found in other species even in mammls
NAM is derivative of NAG, has extra carboxyl ground to link N-terminus of wall peptide
Only seen in bacteria
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Wall Peptides
Use unusual D-amino acids
Protection against enzymes that target L-AA
Have 2 D-Ala at C-terminus
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Transglycosylation
Wall glycans polymerized by tranglycosylation forming glycosidic bonds
Transglycosylase catalyzes the reaction
Glycan stand alternates between NAG and NAM
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Transpeptidation
Reactions links wall peptides together forming net structures
3rd AA usually lysine or mDAP has free amino group
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Transpeptidation Steps
1. C terminal D-Ala is removed by transpeptidase
2. Transpeptidase links 3rd AA to newly liberated C-terminus of first wall peptide to form amide bond
2 Glycan chains linked by transpeptidation between each of their 3 subunits
Futher crosslinkage can occur to make large mesh or last D-ala is cleaved by carboxylase
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Enzymes active on PG
Carboxypeptidase cleaves 5th D-ALA but does not form transpeptide bond
Regulates the degree of cross-linking
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Wall peptides
Show subtle variation between species
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Structural difference between G- and G+ PG
G- has mDAP and G+ has L-Lys
mDAP is Lysine but with an additional Carboxylate group
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Types of Wall peptide cross linking
G- crosslink with mDAP amide bonding to 4th D-Ala of adjacent wall peptide
G+ crosslink with 3rd Lys to crossbridge of 3 or more AA to 4th D-Ala of adjacent wall peptide
G+ cell wall precursors ofter have additional interbridges attached to lysine
S. Aureus has 5 Gly AA in crossbridge
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Unusual variants in Cross bridges
Micrococcus Luteus has cross bridge derived from wall peptide cleaved off from existing cell wall subunit
Crossbrdige terapeptide is cleaved from NAM by N-acetyl-muramyl-L-Alanine amidase
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An Odd case
Corynebacterium poinsettiae has homoserine instead of Lys or mDAP
2nd D-Ala is used to crosslink with D-ornithine used as crossbridge
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Steps to Cell Wall synthesis in S. aureus
1. Cell wall precursous made in cytoplasm where GlcNAc (NAG) is attached to UTP
2. Enzyme converts UDP-NAG to UDP-NAM (MurNAc). AA are added to make wall peptide
3. NAM-wall peptide transferred to lipid and flipped outside of cell membrane to assembly into cell wall
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Cell Division and PG Biosynthesis
Without PG cells cannot properly divide
Many drugs target PG as a result
Some drugs target D-alanine
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Lytic Transglycosylases,N-acetylmuramidase:
Breaks bond between NAM and NAG at reducing end of NAM
Allows new Disaccharide pentpeptide unites into growing glycan strand
Lysozyme: N-acetylmuramidase in aminals (tears and egg white) use to kill bacteria by attacking PG
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Lytic Transglycosylases,N-acetylglucosaminidase
Breaks bond between NAM and NAG at reducing end of NAG
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Other enzymes active on PG
Glucosaminidase (G): Breaks GN-MN
Muramidase (M) : Breaks MN-GN
Amidase (A): Breaks 1st L-ala from MN
11 Hydolase (psi): breaks 4th D-ala from Gly bridge
Lysostaphin (L): Breaks Gly-Gly in interbridge
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Muramidases and Glucosaminidases
Both release PG subunits
Muramidase breaks at NAM reducing end (MN-GN)
Glucosaminidases breaks at NAG reducing end (GN-MN)
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Transpetidase
Cleaves 5th D-Ala and forms bond between 4th D-Ala carboxyl group to Amino group of DAP (G-) or cross bridge (G+)
Energy from cleaved D-Ala is used to from new bond
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Endopeptidases
Hydrolyze wall peptides that link glycan strands
let new PG units to be inserted with cell grows or divides
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N-acetylmuramyl L-alanin amidase
Breaks bond between NAM and 1st L-ala to yield free tetrapeptide subunits
Used for making type-3 PG bridge
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Autolytic enzymes (autolysins)
Causes breakdown of PG leading to cell lysis when uncontrolled
Includes muramidases, glucosaminidases, endopeptidases and amidases
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Bacterial cell shape
ratio of transpeptidation and carboxypeptidation controls crosslinking
Helps determine 3D structure of cell wall and shape of bacteria
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Gram-Staining
Insoluble crystal violet-iodine complex formed inside cell
Alcohol extracts complex from G- but not G+
G+ have thick cell walls with several layers of PG
Alcohol dehydrates layer closing the pores in the wall preventing complex to escape
In G- alcohol penetrates cell wall and extracts complex
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