Monday Article #34: A delve into the molecular biology of the Escherichia coli heat shock response

- How do E.coli survive in times of thermal stress? Let’s find out!-


Introduction


The heat shock response (HSR), which is observed in all living organisms, has long been a research interest of molecular & microbiologists worldwide. This response was first described by the Italian geneticist Ritossa where he noticed a ‘puffing pattern’ in response to thermal shock in the fruit fly Drosophila melanogaster (Ritossa, 1962). The HSR mechanism involves the synthesis of heat shock proteins (HSP) as a result of gene expression in response to hostile conditions (Arsène, Tomoyasu and Bukau, 2000) and as a survival mechanism, needs to be strictly regulated at both the transcriptional and translational levels. In this essay, which will use the prokaryote Escherichia coli as the model organism, will discuss what the HSR is, followed by a description on heat shock proteins (HSP) before focusing on the specifics of transcriptional regulation of HSR and highlighting the importance of such a response to exist for the survival of E. coli at high temperatures.

HSP functions: GroEL-GroES chaperonin system and proteolytic degradation

The heat shock response is activated by different stimuli such as thermal stress or infections. This essay will closely investigate HSR in regards to heat stress induction (30- 42°C) that will lead to the formation of misfolded proteins. This will trigger the expression of HSPs in the form of molecular chaperone proteins to refold these proteins into its once mature and active form. Examples of HSPs in E.coli include DnaK, DnaJ, GrpE, GroEL & GroES (Arsène, Tomoyasu and Bukau, 2000). However, if cellular proteins are unable to be folded by the chaperones, HSPs, in the form of proteases, are able to degrade the impaired protein, firstly, via the ubiquitination process in which the target protein is ubiquitinated, followed by degradation. It is also important to note that HSPs are also produced during normal cellular conditions. FtsH is an example of one such protease (Roncarati and Scarlato, 2017).


A detailed look at the GroEL-GroES system will illustrate that the misfolded protein will be encapsulated within its barrel-shaped three-dimensional complex. DnaJ-DnaK chaperones will transport the misfolded protein to GroEL.GroEL will bind to the exposed hydrophobic regions of the protein. Next, the binding of ATP will induce a conformational change and GroES is recruited to form the cap of GroEL and refolding will proceed. Another ATP molecule will then bind to the opposite side of the complex, releasing both the GroES and the protein molecule back into the cytoplasm (Lennarz and Lane, 2013) (Figure 1). The protease FtsH is tasked with the elimination of compromised proteins. Some of these proteases are able to be reorganised into larger subunits capable of both chaperone and protease action. The proteases also utilise ATP to reorganise polypeptide substrates and transport them to be catabolised by the proteasome (Missiakas et al.1996; Wawrzynow, Banecki and Zylicz, 1996).


Control of HSR at the transcriptional level by σ32 The HSR is both positively and negatively regulated. Positive regulation involves the specialised sigma (σ) factor 32, the gene product of the rpoH gene which binds to the RNA polymerase (RNAP) to form a holoenzyme, thus initiating transcription of the heat shock genes (HSG). σ 32 is the heat shock or transcription factor which enhances transcription (Arsène, Tomoyasu and Bukau, 2000) (Figure 2). σ 32 binds to the 13-15 bp long consensus sequence between the -35 region CCCTTGAA and -10 region CCCGATNT (Arsène, Tomoyasu and Bukau, 2000, Roncarati and Scarlato, 2017). This consensus sequence is different from the -10 TATA sequence. Interestingly, the conserved σ 32 and σ 70 regions of 2.4 and 4.2 are closely linked to the recognition of the aforementioned consensus regions whereby different sets of amino acid residues bind to the -35 element and the -10 downstream element (Kourennaia, Tsujikawa and Dehaseth, 2005). This supports a 1984 paper by Grossman, Erickson and Gross which showed that the DNA-binding domains of σ 32 was able to bind specifically to RNAP and thus, recognising the heat shock promoters without the intervention of the vegetative housekeeping σ 70 through an in vitro transcriptional assay (Grossman, Erickson and Gross, 1984, Kumar et al.1995). It is worth noting that housekeeping σ 70 and σ E factors are involved with promoter activation (P1-5) and transcription of the rpoH gene (Arsène, Tomoyasu and Bukau, 2000) (Figure 2). σ 32 will dissociate and be degraded if the conditions in the cell revert to normal (Lim et al. 2013).

Interestingly, the conserved σ 32 and σ 70 regions of 2.4 and 4.2 are closely linked to the recognition of the aforementioned consensus regions whereby different sets of amino acid residues bind to the -35 element and the -10 downstream element (Kourennaia, Tsujikawa and Dehaseth, 2005). This supports a 1984 paper by Grossman, Erickson and Gross which showed that the DNA-binding domains of σ 32 was able to bind specifically to RNAP and thus, recognising the heat shock promoters without the intervention of the vegetative housekeeping σ 70 through an in vitro transcriptional assay (Grossman, Erickson and Gross, 1984, Kumar et al.1995). It is worth noting that housekeeping σ 70 and σ E factors are involved with promoter activation (P1-5) and transcription of the rpoH gene (Arsène, Tomoyasu and Bukau, 2000) (Figure 2). σ 32 will dissociate and be degraded if the conditions in the cell revert to normal (Lim et al. 2013).

Heat shock genes are also negatively controlled by repressors. These heat shock repressors are able to bind to the operator region, blocking RNAP action and therefore halting transcription when E.coli is not under stress. There will be no gene expression of HSG and hence, no HSPs will be produced. In times of a rise in temperature, there will be induced transcription (Briat, Gilman and Chamberlin, 1985). The repressors are also able to block their own promoters during the negative autoregulation process (Alon, 2007). Examples of E.coli heat respressors include HrcA and HspR. The conserved IR regions of CIRCE are bound by the HrcA repressor during heat stress. The chaperonin GroEL-GroES system initiates DNA-binding when interacting with the HrcA repressor (Roncarati and Scarlato, 2017) (Panel A, Figure 3). Similarly, the HspR represses transcription by binding to conserved HAIR sequences (Panel B, Figure 3). Chaperone DnaK is a known corepressor of HspR. It is also crucial to point out that a build-up of unfolded proteins negatively modulates both HrcA and HspR-DnaK activity.


Why and how this response aids in survival in times of heat stress


It is vital for E.coli to possess this crucial mechanism in order to survive thermal stress. Other than survival, a bacteria’s goal would also be reproduction to form a stable and thriving population. This is extremely important for food-borne pathogenic E.coli variants such as the enterohemorrhagic E.coli O157 which caused the summer 2000 New Deer Outbreak in Aberdeenshire. It was found that this specific E.coli variant was able to survive in soil for 15 weeks as the percentage of positive E.coli O157 samples remained fairly constant from May until July (Pennington, 2014) (Figure 4). This was evidence that O157 was very comfortable in soils during the summer months as they would be able to survive in these warmer conditions by utilising the HSR mechanism.

Conclusion To conclude, the HSR is an important fundamental mechanism required by living organisms such as the E.coli bacteria. It is evident that living organisms, especially in our modern environment will not be able to survive were it not for the existence of the HSR as a key evolutionary protective mechanism. The induction of the molecular chaperones DnaK and the GroEL-GroES system helps the cell to respond appropriately to these changes in the environment. FtsH plays a key role in maintenance of cellular function as to mediate protein degradation to prevent accumulation of non-functional proteins. It was also discussed that ATP is required for these chaperones to perform their functions. We also considered the different strategies used by E.coli to positively and negatively regulate the heat shock genes at the level of transcription. σ32 , the gene product of the rpoH gene is the key player in positive transcriptional control whereby it enhances transcription via protein-protein interactions with the RNAP promoter. It was also stressed that HSPs are usually produced at a moderate amount during normal conditions and that σ32 will be degraded when it is not required. Heat shock is also actively negatively regulated by intervention of repressor proteins HrcA and HspR-DnaK in which their DNA-binding domains will bind to conserved operator regions that are specific to the repressor. It was then highlighted that an aggregation of unfolded proteins is linked to negative modulation of the repressors. We then viewed the significance of the HSR by delving into a case study on the virulent E.coli O157 which is responsible for many outbreaks globally, with the New Deer Outbreak of 2000 being one of them. The data strongly supports the use of the HSR mechanism in ensuring that the pathogenic O157 strain was able to spread and survive for an extended period of time. These encounters with virulent E.coli strains and an understanding of the HSR will aid food microbiologists in providing the best guidance possible to governments, food regulatory authorities and the public in hopes of reducing the number of outbreaks and ensuring food safety for consumers. By developing an understanding of this mechanism, we are able to conclude that thermal killing will not have much of an impact on E.coli due to the presence of HSR. The HSR mechanism is a widely studied area of interest, especially in the model organism E.coli in hopes of unlocking the secrets of improving human health by also applying this useful piece of information to combat other food-borne diseases! REFERENCE(s):

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This article was prepared by Eldrian Tho

 

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