L3 - Life Under Extreme Conditions

Question 1

Give the two ways in which microbes can deal with extreme environments

Answer 1

• Short-term stress response
• Adaptation to environment (e.g. extremophiles, often unable to grow under ‘normal’ conditions)

Question 2

Name the 4 types of extremophiles based on heat

Answer 2

• Psychrophile (-20 - +10˚C)
• Mesophile (20 - 40˚C)
• Thermophile (50 - 80˚C)
• Hyperthermophile (80 - 120˚C)

Question 3

Give the biggest challenges faced my thermophiles

Give an example of a hyperthermophile

Answer 3

Enzymes: maintaining activity

Nucleic Acids: Replication, transcription, translation

Membranes: maintaining correct fluidity

• E.g. Pyrolobus fumarii (up to 121˚C) near black smokers - Archaea

Question 4

In which kingdoms are extremophiles found?

Answer 4

• Bacteria
• Archaea

Question 5

Give an example of a mesophile that has a short term stress response and outline the mechanism

Answer 5

• E. coli, 30˚C to 42˚C = heat shock
• Heat shock induces expression of Heat Shock Regulon (HSR)
• HSR inc. genes encoding chaperone proteins, proteins for FeS protein assembly, DNA repair enzymes
• HSR expression controlled through alternative sigma factor σ32

Question 6

Why is it important to regulate σ32 levels

Answer 6

• To function, σ32 displaces σ70.
• Too much displacement is bad and can cause cell death
• Important to regulate well!

Question 7

Describe the active and inactive forms of σ32

Answer 7

Inactive form: σ32 bound to chaperones DnaK/J and chaperonins GroEL/ES

Active form: σ32 free, replaces σ70 on RNAP, stimulates HSR transcription

Question 8

Describe how σ32 levels are controlled

Answer 8

1) mRNA encoding σ32 forms stem loops including AUG start codon + SD sequence. Loops melt at high temp. causing increase in σ32 translation

2) Mis-folded proteins (from heat) displace σ32 from chaperones = active

3) σ32 has short half life < 1 min. Degraded by FtsH. Degradation suspended under heat shock. Quickly resumed after

Question 9

Describe how Archaea cell membranes can allow survival in extremophilic conditions

Answer 9

• Thermophilic and acidophilic Archaea contain tetra-ether lipids in membrane
• Form rigid monolayer membrane (covalent bonds)
• More stable than ester bilayer lipids in bacteria + eukaryotes (molecular interaction)
• But hard to maintain viable p.m.f

Question 10

Give four points for how thermophilic enzymes remain thermally stable compared to mesophilic counterparts

Answer 10

• More compact, often oligomers
• Greater surface complementarity at dimer surfaces
• Increased no. of stabilising interaction e.g. ion pairs and S-S bonds
• Decreased flexibility

Question 11

Describe factors that differentiate thermophilic proteins from mesophilic proteins using an example

Sketch the relevant diagram

Answer 11

• E.g. enzyme cytochrome C from mesophile P.aeruginosa and thermophile Hydrogenobacter thermophilus
• Similar structure in both
• Denaturation temp. of 47˚C vs 87˚C

Differences:
- Increased hydrophobic interaction at protein centre in thermophile
- Closer packed side chains
- Site-directed mutagenesis confimed these interactions were responsible (expand + see graph pg 20)

Question 12

Outline the mechanisms that can protect against ionising radiation

Describe this through an example. List 5 mechanisms

What is the potential of this example?

Answer 12

• Bacterium D. radiodurans - up to 5000 Gray
• Increased genome copy number
• Highly compacted nucleoid

Efficient repair of double stranded breaks in DNA (lots of relevant proteins):

1) Non - homologous end joining (NHEJ). PprA binds to double stranded DNA, stimulating ligase activity

2) Homologous recombination via RecA

3) DNA end protection by DdrA, binds to 3’ ends + protects against nuclease action

• Potential bioremediation in contaminated sites