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Phys-Pro - Section 1 > Gram - cell envelope > Flashcards

Gram - cell envelope Flashcards

1
Q

What is the primary function of the outer membrane

A

Mainly acts a selective permeability barrier

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

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2
Q

The outer membrane allows passage of hydrophilic molecules through __

A

Porins

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3
Q

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

A

Negative

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4
Q

What kind of receptors are on the outer membrane

A

Phage receptors

O-antigen and OmpA

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5
Q

4 general features of the outer membrane

A
  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
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6
Q

Lipopolysaccharides (LPS) - general features

A

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
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7
Q

LPS - functions

A
  1. Confer negative charge
  2. repel hydrophobic molecules
  3. Repel fat soluble molecules like bile which can harm the bacteria
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8
Q

3 parts of LPS

A
  1. Lipid A
  2. Core polysaccharide/oligosaccharide
  3. O-specific polysaccharide (O-antigen)
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9
Q

Lipid A - structure

A

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

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10
Q

C14 hydroxymyristic acid is only found in which membrane

A

Only the outer membrane

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11
Q

Core oligosaccharide - structure

A

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

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12
Q

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

A

KDO

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13
Q

O-antigen structure

A

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

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14
Q

LPS without O-antigen is referred to as?

A

Rough

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15
Q

LPS with O-antigen is referred to as?

A

Smooth

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16
Q

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

A

Partial loss of virulence

-more susceptible to phagocytosis and serum bactiericidal reactions

17
Q

What does “deep rough” mutants refer to

A

Loss of the more proximal parts of the O-antigen

18
Q

What are deep rough mutants susceptible to?

A

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

19
Q

Explain why Lipid A is toxic

A

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

20
Q

The 3 components of LPS are synthesized ____ of each other

A

Independently

21
Q

Where is Lipid A derived from

A

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

22
Q

Core oligosaccharide synthesis is thought to include

A

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

23
Q

6 steps of O-antigen biosynthesis and complete LPS formation

A

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.

24
Q

Porins - basic structure and distribution

A

Major proteins of the outer membrane

Most contain 3 identical units

25
Q

Porins: mechanism

A

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

26
Q

3 major porins of E. coli

A

OmpC, OmpF, and PhoE

*OmpC is smaller than OmpF

27
Q

When is PhoE produced

A

PhoE is only produced under phsophate limiting conditions

28
Q

When is OmpC present in higher concentrations?

A

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

29
Q

Porins are made up of which secondary structure?

A

Beta sheets linked together by beta turns

The sheets lie in an antiparallel fashion and form a cylindrical tube called the beta barrel

30
Q

What is special about the composition of the beta sheets

A

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

31
Q

What is the function of the eyelet

A

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

32
Q

What is a possible method of antibiotic resistance involving porins

A

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

33
Q

Braun’s lipoprotein function

A

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

34
Q

Braun’s lipoprotein mechanism of action

A

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.

35
Q

Periplasmic space

A

space between the outer and the cytoplasmic membranes

36
Q

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

A

Oxidizing environment

  • allows for the formation of disulphide bonds
  • cytoplasm has a reducing environment
37
Q

7 periplasmic specific proteins

A
  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
38
Q

3 methods of outer membrane mediated antimicrobial resistance

A
  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

Phys-Pro - Section 1 (8 decks)

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