Lecture 3

Question 1

Gram Stain

Answer 1

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-

Question 2

Steps to Gram staining

Answer 2

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

Question 3

Membrane of G+ and G- Bacteria

Answer 3

G+ have PG and cytoplasmic membrane

G- are like G+ except they have a outer membrane made of Lipopolysaccharide and protein

Question 4

Peptidoglycan (PG, Murein)

Answer 4

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

Question 5

PG and osmolarity

Answer 5

PG makes cell wall rigid
Protects bacteria from external osmolarity changes
Digest PG with lysozyme removes protection and cells dies from osmolarity change

Question 6

Sacculus

Answer 6

PG sack retains shape after bacteria is disturbed

Question 7

Structure of PG

Answer 7

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+

Question 8

Wall Glycan

Answer 8

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

Question 9

Wall Peptides

Answer 9

Use unusual D-amino acids
Protection against enzymes that target L-AA
Have 2 D-Ala at C-terminus

Question 10

Transglycosylation

Answer 10

Wall glycans polymerized by tranglycosylation forming glycosidic bonds
Transglycosylase catalyzes the reaction
Glycan stand alternates between NAG and NAM

Question 11

Transpeptidation

Answer 11

Reactions links wall peptides together forming net structures
3rd AA usually lysine or mDAP has free amino group

Question 12

Transpeptidation Steps

Answer 12

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

Question 13

Enzymes active on PG

Answer 13

Carboxypeptidase cleaves 5th D-ALA but does not form transpeptide bond
Regulates the degree of cross-linking

Question 14

Wall peptides

Answer 14

Show subtle variation between species

Question 15

Structural difference between G- and G+ PG

Answer 15

G- has mDAP and G+ has L-Lys

mDAP is Lysine but with an additional Carboxylate group

Question 16

Types of Wall peptide cross linking

Answer 16

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

Question 17

Unusual variants in Cross bridges

Answer 17

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

Question 18

An Odd case

Answer 18

Corynebacterium poinsettiae has homoserine instead of Lys or mDAP
2nd D-Ala is used to crosslink with D-ornithine used as crossbridge

Question 19

Steps to Cell Wall synthesis in S. aureus

Answer 19

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

Question 20

Cell Division and PG Biosynthesis

Answer 20

Without PG cells cannot properly divide
Many drugs target PG as a result
Some drugs target D-alanine

Question 21

Lytic Transglycosylases,N-acetylmuramidase:

Answer 21

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

Question 22

Lytic Transglycosylases,N-acetylglucosaminidase

Answer 22

Breaks bond between NAM and NAG at reducing end of NAG

Question 23

Other enzymes active on PG

Answer 23

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

Question 24

Muramidases and Glucosaminidases

Answer 24

Both release PG subunits
Muramidase breaks at NAM reducing end (MN-GN)
Glucosaminidases breaks at NAG reducing end (GN-MN)

Question 25

Transpetidase

Answer 25

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

Question 26

Endopeptidases

Answer 26

Hydrolyze wall peptides that link glycan strands | let new PG units to be inserted with cell grows or divides

Question 27

N-acetylmuramyl L-alanin amidase

Answer 27

Breaks bond between NAM and 1st L-ala to yield free tetrapeptide subunits Used for making type-3 PG bridge

Question 28

Autolytic enzymes (autolysins)

Answer 28

Causes breakdown of PG leading to cell lysis when uncontrolled Includes muramidases, glucosaminidases, endopeptidases and amidases

Question 29

Bacterial cell shape

Answer 29

ratio of transpeptidation and carboxypeptidation controls crosslinking Helps determine 3D structure of cell wall and shape of bacteria

Question 30

Gram-Staining

Answer 30

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

Question 31

PG of G+

Answer 31

serves as attachment sites for other molecules including protein