Lecture 5. Lipopolysaccharide, Periplasm And Flagella

Question 1

What is lipopolysaccharide (LPS)?

Answer 1

Large complex molecules containing lipid and
carbohydrate (lipid A, core polysaccharide, O side chain)

Question 2

What is the best described form of LPS?

Answer 2

Salmonella
E. coli LPS is nearly identical and also well studied

Question 3

What is LPS known as when free in host?

Answer 3

Endotoxin

Question 4

What makes up lipid A?

Answer 4

Two glucosamine residues linked to fatty acids and
phosphate (occasionally pyrophosphate)

Question 5

What makes up the core polysaccharide?

Answer 5

In Salmonella, 10 (mostly) unusual sugar residues: glucose; galactose; heptulose (x2); 2-keto-3-deoxyoctonate (x3)
Side chains of NAG, phosphate and ethanolamine

Question 6

What is the O side chain?

Answer 6

Variable region, responsible for antigenic make-up of bacteria
Lipid A and core polysaccharide are straight and O side chain flexible and bent
Highly variable composition
Rough (R)/smooth (S) variants depend on chain length

Question 7

What are the functions of LPS?

Answer 7

Lipid A stabilises outer membrane structure
Core polysaccharide charged
Charged, hydrophilic external layer reduces
permeability of hydrophobic substances
Protects against host defences

Question 8

What are endotoxins?

Answer 8

Released during cell division or by lysis of bacterial cells
Can act to prime immune system against a pathogen
If in the blood LPS can cause septic shock syndrome

Question 9

What is the limulus amaebocyte lysate (LAL) test?

Answer 9

An approved procedure for assaying for LPS
Amaebocytes are the blood cells of Limulus polymephus
Contains a clotting factor that is released if coming into contact with bacterial endotoxin
Natural immune mechanism that contains bacterial infection

Question 10

What are important properties of endotoxins?

Answer 10

Heat stable
Toxic in nanogram amounts
Interacts with innate immune system cells
Can result in inflammation, fever, vasodilation, thrombosis, acute disseminated intravascular coagulation, depletion of platelets/clotting factors leading to internal bleeding (haemorrhage), shock, sometimes death

Question 11

Why is the outer membrane more permeable than the inner?

Answer 11

Due to presence of porins
Porins are protein channels that permit passage of small molecules up to around 600 Da

Question 12

What are porins?

Answer 12

Homotrimeric, transmembrane proteins
Highly conserved structure
Form water filled channels in OM

Question 13

Are most porins selective or non-selective?

Answer 13

Most are non-specific channels that allow passage of small (<600 Da) hydrophilic molecules (Most porins are slightly cation selective)
Some porins are selective

Question 14

What is common across all porins?

Answer 14

Unusual structure consisting of a 16-stranded antiparallel β-barrel
Exceptionally stable

Question 15

What can be found in a cross-section of a porin monomer?

Answer 15

Hourglass shape with a central constriction
Hydrophobic band of 25 Å that sits in membrane
Charges inside pore define size of solute that can traverse the channel

Question 16

What do compounds diffuse through porins into?

Answer 16

The periplasm

Question 17

What is the periplasm?

Answer 17

The space between outer membrane and cytoplasmic membrane (Ranges in size from 1 to 70 nm and can be up to 40% of cell volume)
Gel like consistency due to abundant proteins
Removal of cell walls without lysing the cells allows study of the proteins and enzymes present in this space

Question 18

What enzyme activity occurs in the periplasm?

Answer 18

Nutrient acquisition
Energy conservation
Some peptidoglycan synthesis enzymes are periplasmic
Periplasmic binding proteins
Chemoreceptors

Question 19

What is the Sec pathway?

Answer 19

Exports nascent polypeptide through cytoplasmic
membrane using a translocase
Folding of protein occurs after translocation
Proteins have an N-terminal signal peptide

Question 20

What is the TAT pathway (twin arginine translocase)?

Answer 20

Exports fully folded enzymes across cytoplasmic membrane
Proteins have a twin arginine in N terminal region

Question 21

What are flagella?

Answer 21

Long thin extracellular helical structures made of protein subunits that aid in motility

Question 22

What is the structure of the flagellum?

Answer 22

Complex ring structures anchored into the membrane and cell wall/outer membrane
Flagellum shaft is easily removed by vigorous shaking
Different antigenic properties of shaft, tip of shaft and hook
The motor is driven due to transfer of protons through the ring structure (from periplasm to cytoplasm)

Question 23

What is the structure of the flagellum filament?

Answer 23

Filament made of a single protein called flagellin
Base is different to the shaft and is known as the hook
Single protein connecting the shaft to the motor
Moves like a rotary motor

Question 24

Where does growth in flagella take place?

Answer 24

At the tip

Question 25

Process of flagella synthesis

Answer 25

1. MS and C rings in cytoplasmic membrane
2. Motor proteins
3. P and L ring, hook and cap
4. Flow of flagellin through hook, growth of filament

Question 26

How does the motor in flagella work?

Answer 26

Rotary motion provided by the basal structures
Powered by proton motive force
Mutational analysis suggests dependence on Asp residue in MotB

Question 27

How are flagella different in Gram +ve than Gram -ve bacteria?

Answer 27

In Gram +ve, no L and P rings
Anchor in the membrane layer is more complex
Mot proteins surround inner ring and movement of these relative to each other provides the force

Question 28

What is the flagellum also known as?

Answer 28

H antigen