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1

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-

2

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

3

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

4

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

5

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

6

Sacculus

PG sack retains shape after bacteria is disturbed

7

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+

8

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

9

Wall Peptides

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

10

Transglycosylation

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

11

Transpeptidation

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

12

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

13

Enzymes active on PG

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

14

Wall peptides

Show subtle variation between species

15

Structural difference between G- and G+ PG

G- has mDAP and G+ has L-Lys
mDAP is Lysine but with an additional Carboxylate group

16

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

17

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

18

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

19

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

20

Cell Division and PG Biosynthesis

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

21

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

22

Lytic Transglycosylases,N-acetylglucosaminidase

Breaks bond between NAM and NAG at reducing end of NAG

23

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

24

Muramidases and Glucosaminidases

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

25

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

26

Endopeptidases

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

27

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

28

Autolytic enzymes (autolysins)

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

29

Bacterial cell shape

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

30

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

31

PG of G+

serves as attachment sites for other molecules including protein