Molecular Biology of the Heat Shock Response in prokaryotes
Prevent E. coli in poultry | MS Schippers
Introduction
To survive in an environment, it is essential to have the capacity of adapting to changes in temperature. This, in unicellular organisms such as prokaryotes, is done on the molecular level, although the first line of defence is in the form of the cellular wall. To cope with high temperature changes prokaryotes have developed various heat-shock response systems such as sigma transcriptional factors and repressors as well as thermosensor elements within RNA and DNA. These mechanisms activate the production of proteins such as chaperone and protease protein groups, that protect against the hazardous environmental conditions and restore homeostasis, but may also trigger pathogenic activation (Klinkert and Narberhaus, 2009).
Although both bacteria and archaea are considered prokaryotic organisms (Brown and Doolittle, 1997), this article will focus in-depth on the bacterial heat-shock responses, excluding the Archaea domain. The heat-shock response and heat shock proteins will be explained. The pathogenic response of E. coli and Shigella flexneri species in response to DNA thermoreceptors will be mentioned. This will be then followed by the heat-shock response of sigma32 factor translation in E. coli and a final conclusion on the bacterial temperature change response.
Bacterial heat shock response
Bacteria are known to survive in various environmental conditions with varying temperatures and have therefore unique coping mechanisms. Although several bacterial species have adapted and thus been observed living in extremely hot environments, all organisms have limited capacity for temperature changes. However, the environment occupied by bacteria does not only change due to the season but with every host colonisation the external environment change for the bacteria. Nonetheless, the heat can have several effects on the organism. Increase in temperature causes protein denaturation and melting of DNA which results in misaligning or bundle formation thus interfering with transcription and translation. It can also result in the loss of structure of lipids due to the interactions between hydrophilic heads and hydrophobic tails that disassociates with increasing temperature. Since lipids make up the cellular membrane, it´s deformation can have adverse results on the survival of the individual. Therefore, to cope with heat-shock the bacterial cell has evolved several responses that are governed by various heat-shock response proteins (Klinkert and Narberhaus, 2009).
There are several ways in which bacteria handle heat-shock, however, all follow a similar pattern to restore its homeostasis. Firstly, the organism recognises the heat through a sensor which then causes repression or induction of adequate genes that trigger the translation of heat-shock proteins. The product itself or the return to optimal temperature then acts as the inhibitor creating negative feedback and restoring homeostasis (Schumann, 2017).
Heat-shock proteins
The heat-shock response of bacteria involves the expression of genes that code for specific heat-shock proteins (HSPs). These proteins include the chaperone and protease groups of proteins that specialise in detecting denatured proteins due to an increase in temperature. These molecules are also involved in protein tracking though endo- and exocytosis as well as cell signalling and receptor topology. DnaK and GroESL are examples of chaperones that are both associated with heat shock protein90. It is present in both prokaryotic as well as eukaryotic organisms with a 50% similarity in sequence and affects the cell´s response to heat shock. For the heat-shock response to occur accurately HSPs are regulated with sigma factors that further aid the response to adverse temperature change (Maleki et al., 2016).
DNA thermosensors
The increase in temperature can influence the DNA structure thereby interfering with the expression of necessary genes. DNA can thus serve as a thermosensors and happens in various ways. In E. coli and Sulfolobus an increase in temperature causes plasmids to positively supercoil, in Clostridium perfringens species´ DNA has been observed to unbend itself at higher temperatures, whereas in the bacterium Shigella flexneri the H-NS binding in the DNA is highly temperature-dependent and thus breaks at higher temperatures exposing genetic information. These change in DNA structure suppresses some gene expressions, however, through structural changes other genes become available for transcription coding for a heat-shock response to protect the bacterium from the heat (Klinkert and Narberhaus, 2009).
Temperature-dependent pathogenic activation
The heat-shock response can activate the pathogenic response as the temperature of the host organism can be higher than the temperature of the original environment. This can be observed in the bacterium Shigella flexneri and E. coli, which are human pathogens that are temperature-dependent. With an increase in temperature from below 32 to 37˚C caused by the invasion of a host, the virF promoter, normally bent and inactive due to the H-NS bond, becomes exposed, serving as a thermosensor. The exposed promoter can be accessed by the RNA polymerase and be transcribed for virF anti-H-NS protein as well as activate the FIS (transcriptional activator) which causes a positive feedback loop increasing the virF transcription, ultimately resulting in the HSR. The virF protein activates the iscA and virB genes which trigger the invasion of the host, thus aiding the survival of the pathogen. This response is controlled with temperature and thus, with a decrease in temperature, the H-NS bond can reform, blocking access to the virF promoter (Di Martino et al., 2016).
Figure 1. The location and temperature-dependent activation of virF gene promoter on the DNA (Falconi et al., 1998).
HSR though Sigma 32 factor activation
One of the heat-shock responses (HRS) observed in the bacteria Escherichia coli involves the sigma32 subunit encoded by the rpoH gene. It is found at low concentrations during low temperatures but increases in amount by 15 folds after 5 minutes with an increase in temperature. Escherichia coli has optimal growth at 37˚C. At this temperature sigma70 is attached to the RNA polymerase triggering gene expression that is essential for cell´s natural growth. At this temperature, σ32 is bound to heat shock proteins called chaperones such as GroESL and DnaK and is thus inactive (Schumann, 2017). An increase in temperature to >40˚C could be lethal to the bacteria. To prevent this, the increase in temperature activates HSR of E. coli. The exposure to this temperature causes disassociation between σ32 and the chaperone either due to the reduction in stability of binding interaction or due to the loss of suppression between the chaperone and σ32. This is because an increase in temperature also results in protein denaturation which is detected by DnaK thus sequestering the chaperone to the denatured protein. This is stronger than the bonding between DnaK and σ32, freeing the sigma factor. At the same time the increase in temperature causes a reduction in the RNA polymerase to σ70 bonding resulting in disassociation. The σ32 can then bind to the RNA polymerase forming a holo-enzyme that binds to the promoters at the CTTGAAA sequence in the -35 region or the CCCCATNT sequence at the -10 region and thereby allows transcription of the genetic information necessary for the HSR (Wang and Dehaseth, 2003). One of the two main proteins expressed are the GroEL and GroES proteins essential for both DNA and RNA synthesis at temperatures of 42˚C (Fayet, Ziegelhoffer and Georgopoulos, 1989).
During the HSR the concentration of sigma32 is constantly regulated by the membrane-bound FtsH protease. It constantly degrades the free-floating σ32, decreasing its concentrations and thereby regulates the HSR (Tomoyasu et al., 1995). Another regulatory system involves the temperature decrease which results in the denatured proteins returning to their original state thus unbinding from DnaK. The free DnaK can then rebind with σ32 also decreasing its accessibility to RNA polymerase through negative feedback and thereby allowing σ70 to bind instead. In this way, the cell can return to its homeostasis (Wang and Dehaseth, 2003).
Figure 2. Heat-shock response through the activation of sigma32 factor; its effect and inhibition in Escherichia coli (Yura and Nakahigashi, 1999).
Conclusion
The HSR in bacterial cells serves as a necessary survival process for the protection of natural cellular functions, however, it can also serve as the triggering point for pathogenic attack. The specific DNA thermosensors and/or chaperones respond to changes in the folding of molecular structures signalling an increase in temperature. In the Shigella flexneri species, the increase of temperature caused though entering the host results in changes to DNA structure exposing genes that further trigger a pathogenic response. This can be also observed in E. coli bacteria, however, this species is also known for its sigma32 factor that, when released from its bonding with chaperone can trigger a response to protect the cellular functions from degrading due to the temperature change. These examples show that temperature can trigger various responses that all have evolved to increase species survival and enhance its growth. This being especially important in bacteria as their environment changes vastly.
The knowledge of molecular processes in bacteria can help understand the evolution of temperature-dependent coping mechanisms and give insights to eukaryotic mechanisms. However, understanding of HSR in bacteria can also be important when it comes to human health and fighting diseases. Therefore, further research on this topic is highly important for the development of medicine.
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