1. Bacterial Physiology (Micr430)
Lecture 17
Bacterial Responses to Environmental Stress
(Text Chapter: 19)

2. Environmental Stress and Response
Bacteria respond in a variety of physiological ways to environmental stresses such as changes in pH, temperature, food source, radiation damages and oxidative toxicity.
We will describe:
Heat-shock response
DNA damage response
SOS response

3. Thermal Stress and Heat Shock Response
Upon a shift from 30º to 42º C, E. coli and other bacteria transiently increase the rate of synthesis of a set of proteins called heat shock proteins (HSPs).
Many of these HSPs are required for cell growth or survival at more elevated temperatures (thermal-tolerance).
Heat shock proteins are classified based on molecular weights
Hsp70 family (DnaK)
Hsp60 family (GroEL)
Hsp40 family (DnaJ)

4. Heat Shock Response
In E. coli, after a temperature upshift (32 ºC to 42 ºC), there is a transient increase in the amount of sigma factor σ32, also called σH or RpoH.
σ32 recognizes promoters of genes in a major heat shock regulon – the σ32 regulon.
During growth at 30 ºC, σ32 can be degraded by several proteases. However, if σ32 is bound to RNAP, it is protected from degradation.

5. Heat Shock Response
Cis-acting mRNA sites within the 5’ region of rpoH (σ32 or σH ) message form temp sensitive secondary structures that sequester the ribosomal binding site.
At higher temperature, these secondary structures melt, thereby enabling more efficient translation of the rpoH message.

6. Heat Shock Response
The cell uses the DnaK-DnaJ-GrpE chaperon team to interact with σ32 at low temperature, sequestering σ32 from RNAP.
Failure to bind RNAP facilitates degradation of the σ32 factor.
Upon heat shock, there is an increase in the number of other unfolded or denatured proteins that can bind to DnaK or DnaJ. This reduces the level of free DnaK/DnaJ molecules available to bind σ32, allowing σ32 to bind RNAP, which protects it from degradation.

7. (From Moat book)

8. Functions of E. coli Hsps
Fold newly synthesized proteins at all temperatures
Export proteins at all temperatures
Refold misfolded polypeptides
Perform proteolysis of improperly folded or otherwise abnormal proteins

9. DNA Damage Repair UV-damaged DNA: by photoreactivation
Damaged DNA: by nucleotide excision repair
Damaged DNA: by base excision repair

10. Repairing UV-damaged DNA
Exposure to UV can cause formation of thymine dimers
During photoreactivation, a special enzyme called DNA photolyase reverses the dimerization reaction upon absorbing blue light.

12. Nucleotide Excision Repair
In E. coli, the products of three genes, uvrA, uvrB and uvrC, encode UvrABC endonuclease
It recognizes distortion in DNA region caused by UV-induced thymine dimers and cuts both sides of the dimer
Gap is filled in by DNA pol I and sealed by DNA ligase.

14. Repair of Deaminated Bases
A common type of DNA damage is the deamination of bases (amino group is replaced by keto group)
Deaminated bases pair with wrong bases during replication, creating mutations
To repair, deaminated bases are removed by DNA glycosylases – catalyzing breakage of the N-glycosyl bond between the base and the sugar, leaving an apyrimidinic or apurinic site. These sites are called AP sites

15. Repair of Deaminated Bases
After the base is removed, AP endonucleases cleave the phosphodiester bond next to the AP site, usually on the 5’ side
This generates 3’-hydroxyl end, which is extended by DNA pol I as the 5’-exonuclease activity of this enzyme removes a portion of damaged DNA ahead.
DNA ligase seals the nick.

17. SOS response
SOS is response to distress signal generated by DNA damage
This results in the induction of over 20 unlinked genes whose products are involved in repairing DNA damages, allowing DNA replication to proceed past the damaged site and stalling the cell division to give the cell time to repair damaged DNA.

18. SOS response
SOS signal is generated when RecA protein binds to ssDNA (produced during DNA damage)
When the signal is received, the SOS regulon is induced by inactivation of a repressor LexA.