Thermal Stress and the Heat Shock Response in Microbes
Types of Microbes on the basis of Temperature
There are three types of microbes on the basis of temperature:
( T 0 °C ).
- Animal and human pathogens
“The stress induced in the body of microbes due to high temperature is called thermal stress.”
Heat Shock Response:
This is a ubiquitous protective and homeostatic cellular response cope with heat-induced damage in proteins. Upon a shift from 30◦ to 42 ◦C bacteria transiently increase the rate of synthesis of a set of proteins called heat shock proteins (HSPs).
Among the induced proteins are:
- DnaJ and DnaK,
- RNA polymerase σ70 subunit (rpoD)
- Lon protease
Nearly 50 heat shock–inducible proteins identified in E. coli.
Types of Heat Shock Responses:
There are two types of heat shock responses:
- Heat shock response against cytoplasmic thermal stress
- Heat shock response against periplasmic thermal stress
Gram-negative cell has two membrane-bound subcellular compartments of different conditions:
Cytoplasm: Cytoplasm is energy rich, reducing and osmotically stable.
Periplasm: While periplasm lacks ATP, oxidizing and in contact with the external milieu.
Optimal cell growth requires that the cell senses and responds to changes in these disparate sub cellular compartments. Stress responses in E. coli and S. typhimurium are compartmentalized into cytoplasmic and extracytoplasmic response
1.Heat Shock Response against Cytoplasmic Thermal Stress
The σᴴ regulon:
The σᴴ regulon provides protection against cytoplasmic thermal stress. The E. coli rpoH locus (htpR) encodes a 32 kDa σ factor, also called σᴴ or σ³², redirects promoter specificity of RNA polymerase. The σᴴ protein regulates the expression of 34 heat shock genes. Heat shock first causes an elevation in σᴴ levels, which in turn increases the expression of sigma H target genes.
Regulation of Sigma H:
Sigma H protein can be regulated at 3 levels
- At translation
- By degradation
- At transcription
Regulation of σᴴ at Translation:
First, a temperature upshifts from 30 ◦ to 42 ◦C results in the increased translation of rpoH message. Cis-acting mRNA sites within the 5’ region of rpoH message form temperature- sensitive secondary structures that sequester the ribosome-binding site. At higher temperatures, these secondary structures melt, thereby enabling more efficient translation of the rpoH message. In addition to the increased translation of rpoH message, the σᴴ protein itself becomes more stable, at least transiently.
Regulation of σᴴ by degradation:
The mechanism regulating proteolysis centers on whether σᴴ associates with RNA polymerase. During growth at 30 ◦C, σᴴ can be degraded by several proteases including FtsH, HslVU, and ClpAP. If σᴴ is bound to RNAP, σᴴ is protected from degradation. The cell uses the DnaK-DnaJ-GrpE chaperones to interact with σᴴ at low temperature, sequestering σᴴ from RNA polymerase. Failure to bind RNAP facilitates degradation of the σ factor.
Upon heat shock, increase in the number of other unfolded or denatured proteins occur that can bind to DnaK or DnaJ. This reduces the level of free DnaK/DnaJ molecules available to bind σᴴ, allowing σᴴ tobind RNAP, which protects σᴴ from degradation. When cell reaches the adaptation phase following heat shock, the levels of DnaK and DnaJ rise (both induced by σᴴ) and can again bind σᴴ, redirecting it toward degradation.
Regulation of σᴴ at transcription:
In addition to translational and proteolytic controls, production of σᴴ is regulated at the transcriptional level via a feedback mechanism. There are four promoters driving rpoH expression. Three of which are dependent on σ70, the housekeeping σ factor. The gene encoding σ70, rpoD, is also a heat shock gene induced by σᴴ. So, increased production of σᴴ increases σ70, which increases transcription of rpoH. The fourth rpoH promoter is recognized by another σ factor, σᴱ, encoded by rpoE.
Environmental Agents Triggereing Heat Shock Response:
The heat shock response is also triggered by a variety of environmental agents as;
- UV irradiation
- Agents that inhibit DNA gyrase.
Induction by all of these stimuli occurs through σᴴ. How can all of these diverse stresses activate rpoH? The only explanation that appears reasonable is the accumulation of denatured or incomplete peptides. There is a potential alarmone that has been implicated in signaling expression of this global network. The molecule is diadenosine 5-, 5-P1, P4-tetraphosphate (AppppA), which is made by some aminoacyl–tRNA synthetases (e.g., lysU) at low tRNA concentrations. How this may influence the response is not known.
2. Heat Shock Response against Periplasmic Thermal Stress
The extra cytoplasmic response pathways involve two partially overlapping signal transduction cascades:
- σᴱ regulation
- Cpx systems
These pathways are induced by accumulation of misfolded proteins in the periplasmas under stress.
σᴱ is a member of the extracytoplasmic function (ECF) subfamily of σ factors.
In E. coli, σᴱ is responsible for the transcription of genes including:
- rpoH (σᴴ)
- degP (htrA) encoding a periplasmic protease for the degradation of misfolded proteins
- fkpA encoding a periplasmic peptidyl prolyl isomerase
- rpoE rseABC operon
Rpo E rseA.B.C operon:
The gene encoding σᴱ, rpoE, is the first member of an operon followed by the genes rseA, B, and C. RseA is a transmembrane protein whose cytoplasmic C-terminal domain interacts with σᴱ, acting as an anti-σfactor. The periplasmic face of RseA binds to the periplasmic RseB.
Extracellular stress in some way signals increased proteolysis of RseA by the periplasmic protease DegS, thus relieving the anti-σ effect of RseA on σE. It has been proposed that Rse Band perhaps other periplasmic proteins involved in protein folding protect RseA from degradation by binding to the RseA periplasmic domain, capping the target site of DegS.
Stress-induced misfolding of periplasmic proteins would titrate the RseA cap proteins off of RseA, rendering the anti-σ factor vulnerable to attack by DegS. The result would be increased activity of σᴱ leading to increased levels of σᴱ protein and RseA anti-σ (since they form an operon). The increased amount of σᴱ will drive further expression of genes whose products handle the periplasmic damage while the of RseA will enable the cell to down regulate the system once the capping proteins are again free to bind and protect RseA from DegS degradation.
A second system dedicated to protecting the periplasmic perimeter of the cell is the CpxRA two-component system with:
- CpxA playing the role of membrane-localized sensor histidine kinase
- CpxR as the cytoplasmicresponse regulator.
CpxA responds to envelope stress by autophosphorylation followed by phosphotransfer to CpxR. CpxR∼P activates expression of those genes that response to heat shock.