Gram - cell envelope

Question 1

What is the primary function of the outer membrane

Answer 1

Mainly acts a selective permeability barrier

-not as permeable to hydrophobic molecules as the cytoplasmic membrane is

Question 2

The outer membrane allows passage of hydrophilic molecules through __

Answer 2

Porins

Question 3

The outer membrane confers what kind of charge on the bacteria?

Answer 3

Negative

Question 4

What kind of receptors are on the outer membrane

Answer 4

Phage receptors

O-antigen and OmpA

Question 5

4 general features of the outer membrane

Answer 5

1. LPS on the outer leaflet
2. Phospholipids form in the inner leaflet
3. Outer membrane proteins (porins)
4. Braun’s lipoprotein which is associated with the peptidoglycan

Question 6

Lipopolysaccharides (LPS) - general features

Answer 6

Also referred to as endotoxin/pyogen

Lipid A is the element containing toxic activity and will illicit a strong immune response in mammals

• minor: cytokine induction
• major: septic shock and hypotension

Question 7

LPS - functions

Answer 7

1. Confer negative charge
2. repel hydrophobic molecules
3. Repel fat soluble molecules like bile which can harm the bacteria

Question 8

3 parts of LPS

Answer 8

1. Lipid A
2. Core polysaccharide/oligosaccharide
3. O-specific polysaccharide (O-antigen)

Question 9

Lipid A - structure

Answer 9

Hydrophilic head made of 2 molecules of NAG phosphates joined by a ß-1,6 glycosidic bond

Has 6 fatty acids linked to the NAGs

4 of the 6 FAs are C14 hydroxymyristic acids
-not present in cytoplasmic membrane phospholipids

Question 10

C14 hydroxymyristic acid is only found in which membrane

Answer 10

Only the outer membrane

Question 11

Core oligosaccharide - structure

Answer 11

Short (~10 residues) between Lipid A and the O-antigen

Composed of 4-5 different sugars

2 unusual sugars present: C8 keto-deoxy-octonic acid (KDO) and C7 heptose.

Generally the core oligosaccharide is the same in most gram neg bacteria

Question 12

Which sugar present in the core polysaccharide is commonly used as a marker for the outer membrane in fractionation experiments?

Answer 12

KDO

Question 13

O-antigen structure

Answer 13

Long polysaccharide chain attached to the core oligosaccharide

Repeating unit of 4 or 5 sugars

Highly variable structure and composition between organisms and even within the same species

Question 14

LPS without O-antigen is referred to as?

Answer 14

Rough

Question 15

LPS with O-antigen is referred to as?

Answer 15

Smooth

Question 16

In E. coli and Salmonella, loss of the O antigen results in:

Answer 16

Partial loss of virulence

-more susceptible to phagocytosis and serum bactiericidal reactions

Question 17

What does “deep rough” mutants refer to

Answer 17

Loss of the more proximal parts of the O-antigen

Question 18

What are deep rough mutants susceptible to?

Answer 18

a range of hydrophobic compounds, including antibiotics, detergents, bile salts and mutagens

Due to the fact that the region lost contains lots of charged groups

Question 19

Explain why Lipid A is toxic

Answer 19

When gram neg. bacterial cells are lysed by the immune system, fragments of the membrane begin to circulate which can cause fever, diarrhea and possible septic shock

Question 20

The 3 components of LPS are synthesized ____ of each other

Answer 20

Independently

Question 21

Where is Lipid A derived from

Answer 21

UDP-NAG via a series of reactions involving the addition of 4 fatty acid (hydroxymyristic) chains and 2 KDO. Two more fatty acid chains are then added

Question 22

Core oligosaccharide synthesis is thought to include

Answer 22

membrane-bound glycosyltransferases, which add one sugar at a time to the growing glycan chain.

Question 23

6 steps of O-antigen biosynthesis and complete LPS formation

Answer 23

1&2: glycosyltransferase enzymes sequentially add sugar residues to the C55 lipid carrier to form the polymer
-same one used in peptidoglycan synthesis

3&4: newly synthesized O antigen is exported across the inner membrane by the ABC transporter
-conformational change in the ABC transporter achieves this

5: O-antigen is ligated to the lipid A-core oligosaccharide by a ligase

6: complete LPS molecule is then translocated across the periplasm and to the outer membrane by the LPS transport proteins
- Lpt A, B, C, etc.

Question 24

Porins - basic structure and distribution

Answer 24

Major proteins of the outer membrane

Most contain 3 identical units

Question 25

Porins: mechanism

Answer 25

Form small hydrophilic channels that allow the entry of low MW (

Question 26

3 major porins of E. coli

Answer 26

OmpC, OmpF, and PhoE *OmpC is smaller than OmpF

Question 27

When is PhoE produced

Answer 27

PhoE is only produced under phsophate limiting conditions

Question 28

When is OmpC present in higher concentrations?

Answer 28

OmpC is present in higher quantities in high osmolarity media and in the intestine (compared to OmpF)

Question 29

Porins are made up of which secondary structure?

Answer 29

Beta sheets linked together by beta turns The sheets lie in an antiparallel fashion and form a cylindrical tube called the beta barrel

Question 30

What is special about the composition of the beta sheets

Answer 30

have alternating polar and nonpolar residues The polar residues face inward and form the lining of the barrel while the NP residues interact with the membrane

Question 31

What is the function of the eyelet

Answer 31

The eyelet is a loop that partially blocks the opening of the channel and defines the size of solute that can traverse the channel

Question 32

What is a possible method of antibiotic resistance involving porins

Answer 32

All antibiotics must pass through porins so by changing the composition of AA facing inside the barrel OR on the eyelet could potentially inhibit this

Question 33

Braun's lipoprotein function

Answer 33

Small but abundant protein that provides a way for the peptidoglycan to attach to the outer membrane

Question 34

Braun's lipoprotein mechanism of action

Answer 34

C-terminal lysine is covalently bonded to diaminopimelic acid molecule on the PG peptide. -3rd position on the tetrapeptide N-terminal cysteine is bonded with two fatty acid chains that are embedded in the inner leaflet of the outer membrane. The N-terminal cysteine residue is part of the lipoprotein motif “LXXC”. During protein secretion, LXX is cleaved and fatty acid chains are added to the cysteine.

Question 35

Periplasmic space

Answer 35

space between the outer and the cytoplasmic membranes

Question 36

The periplasmic space has a ____ environment, which differs from the cytoplasm

Answer 36

Oxidizing environment - allows for the formation of disulphide bonds - cytoplasm has a reducing environment

Question 37

7 periplasmic specific proteins

Answer 37

1. Hydrolytic enzymes involved in processing nutrients 2. Nutrient binding proteins that bind and deliver to specific transporters in the cell membrane 3. Biosynthetic enzymes (like those for peptidoglycan synthesis) 4. Proteins involved in fitness and stress survival 5. Cytochrome Cs that oxidize carbon or inorganic compounds and deliver electrons to the ETC 6. Detoxifying agents like ß-lactamase that degrade penicillins 7. TonB protein used to transport solutes unable to diffuse through porins

Question 38

3 methods of outer membrane mediated antimicrobial resistance

Answer 38

1. Down regulation of porin channels 2. Slow the rate at which antibiotic enter the cell by mutating the amino acid residues of the ß barrel 3. Periplasmic proteins - ß-lactamases that can degrade penicillins