In the subsequent years, several heat-shock repressor proteins were identified and their mechanisms of regulation via interaction with conserved DNA elements located in the promoters of heat-shock genes were characterized.
A common feature of heat-shock repressors is, besides the negative regulation of target genes, that they repress their own promoter. Consequently, upon heat stress and subsequent rapid induction of the controlled promoters, repressor concentration rapidly increases and reaches a threshold amount necessary for the re-establishment of the repressed state. In this way, the induction phase that rapidly appears upon heat stress is immediately followed by a shut-off phase of transcription.
Currently, it appears well established that negative transcriptional regulation through dedicated repressors constitutes an important and widespread mechanism employed by bacteria to control heat-shock gene expression, not necessarily as an alternative to positive regulation described above. The observation that a conserved sequence element is embodied in the promoter regions controlling the transcription of class I heat-shock genes of B. Specifically, an inverted repeat IR consisting of 9 bp separated by a 9-bp spacer was identified in the DNA region close to the transcription start site of dnaK and g roESL genes Schmidt et al.
The role of this IR as a negative cis -element in the control of chaperone expression was demonstrated by introducing mutational changes within the upstream, downstream or both halves of the IR preceding dnaK and observing increased expression at low temperature and a reduction in the stimulation of the operon after heat-shock Zuber and Schumann Considering that the conserved IR was observed in many different bacterial species, always within promoters controlling chaperone genes, it was named CIRCE for c ontrolling i nverted r epeat of c haperone e xpression.
Even though the possibility was initially considered that the CIRCE sequence could act alone through the formation of a temperature-dependent secondary structure, the demonstration of involvement of a DNA-binding protein in CIRCE-dependent negative regulation followed soon after. In particular, the inactivation of orf39 , the first gene of the B. Moreover, the characterization of regulatory mutants constitutively overexpressing groE and dnaK at low temperature revealed that all mutations map within orf The finding that cell extracts of E.
Similar observations were made for Caulobacter crescentus , where disruption of an orf39 homologous gene led to the conclusion that it encoded a negative regulator and, thus, it was named HrcA for heat-shock r egulation at C IRCE elements Roberts et al. Detailed biochemical studies aimed at the molecular level dissection of the HrcA-CIRCE interaction have been hampered in many cases by its instability and strong tendency to form insoluble aggregates.
For this reason, HrcA binding to target promoters has been characterized in detail only in a limited number of cases. On both target promoters, the protected regions cover about 30 bp, suggesting that a dimer of HrcA interacts with the DNA. In this latter microorganism, in vitro transcription assays of selected promoters carried out in the presence of different concentrations of purified HrcA demonstrated that it was able to specifically repress dnaK transcription in a concentration-dependent manner Wilson and Tan The position of the CIRCE element is almost always in a region close to the transcription start site, overlapping the portion of the DNA contacted by the RNA polymerase during initiation of transcription.
The mechanism of repression exerted by HrcA upon binding to the CIRCE element relies on the physical occupancy of the core promoter with a consequent impediment of RNA polymerase binding under physiological growth conditions Fig. HrcA-mediated repression of heat-shock genes is generally responsive to different kinds of environmental stresses.
In many cases, when bacterial cells are exposed to stressful conditions such as high temperature, high salt concentration or a condition that provokes accumulation of misfolded proteins in the cytoplasm, the transcription from HrcA-controlled promoters is rapidly derepressed Schmidt, Hertel and Hammes ; Laport et al. To date, the crystal structure of the HrcA repressor of the hyperthermophile Thermotoga maritima provides the only detailed structural information for this heat-shock regulator.
The 2. The detailed structure of the DBD, combined with results of mutagenesis studies of conserved residues Hitomi et al. It is worth noting that the amino acid sequence similarity of HrcA proteins from different species is surprisingly low. Hence, the molecular features derived from the solved structure described above may be different among the various HrcA regulators. In order to identify additional genes regulated by HrcA besides the typical groE and dnaK genes, genome-wide expression studies have been combined with the search of conserved CIRCE elements and with in vitro DNA-binding studies.
What generally emerges from such approaches is that HrcA is involved, directly or indirectly, in the regulation of several genes linked to various cellular processes. For example, a microarray-based transcriptome analysis performed in Listeria monocytogenes revealed that HrcA regulates, albeit indirectly, genes involved in stress response, metabolism, translation and DNA replication Yuewei et al. Similar pleiotropic effects of hrcA mutation were observed with analogous approaches in other microorganisms, including H.
However, the number of genes directly regulated by HrcA appears to be restricted and limited to genes involved in stress response. For example, in the human pathogen Mycoplasma genitalium , the genes encoding the stress proteases Lon and ClpB possess a conserved CIRCE element within their promoter regions, suggesting a HrcA-mediated negative regulation Musatovova, Dhandayuthapani and Baseman This GroE-mediated feedback control of HrcA activity is negatively modulated by accumulation of unfolded proteins during stress.
B HspR-mediated repression of heat-shock genes at normal growth temperature is due to binding of the regulator to conserved HAIR sequences close to the core promoter, thereby interfering with RNA polymerase binding indicated by a black arrowhead. The HspR-DnaK functional interaction is negatively modulated by the accumulation of misfolded proteins in the cytoplasm that titrate away the chaperone.
C Current model for the mechanism of repression and for the heat-dependent regulation of CtsR repressor activity. During control conditions, CtsR binds to conserved IR sequences and represses transcription of heat-shock genes depicted by a black arrowhead.
Following a sudden increase of temperature, CtsR undergoes a heat-induced structural alteration and loses affinity for DNA indicated by a gray dashed line. Upon dephosphorylation by the cognate phosphatase YwlE, McsB becomes deactivated. The heat-shock transcriptional repressor HspR was first discovered and characterized in the Streptomyces genus. Specifically, it was shown that, in Streptomyces coelicolor , the distal gene of the heat-inducible dnaK operon encoded a novel heat-shock protein, similar to the GlnR repressors of Bacillus spp.
This strongly suggests a direct role for HspR in heat-shock gene regulation Bucca et al. Genetic and biochemical evidence of HspR regulatory function followed soon after. In particular, it was shown that disruption of the hspR gene led to high-level constitutive transcription of dnaK operon.
Moreover, the addition of an E. Similar observations were made in the same year in S. Genes similar to hspR were observed in several other bacterial species, and HspR-binding sites were detected upstream of various heat-shock genes of these microorganisms.
So far, HspR repressors have been found and characterized, to various extents, in several bacterial species and it is now well established that this represents, in addition to the widespread HrcA repressor, a widely employed system to negatively control heat-shock genes transcription. This observation suggests that HspR prevents RNA polymerase binding at physiological temperature by a steric hindrance mechanism at the promoter Fig. In some cases, however, HspR-HAIR interaction takes place far upstream of the core promoter region, in an atypical position for a transcriptional repressor Spohn et al.
Another intriguing example of complex HspR-mediated regulation has been observed in Mycobacterium tuberculosis. Moreover, a direct HspR-PhoP interaction has been demonstrated, as well as the crucial importance of the simultaneous presence of both regulators for heat-shock-dependent in vivo regulation of acr2 Singh et al. Accordingly, the current model for acr2 regulation postulates that under normal growth condition both the PhoP and HspR, bound to their target sites, form a higher-order DNA-protein structure that prevents RNA polymerase binding and the direct HspR-PhoP interaction provides additional stability to this complex.
Initially identified as the regulator of major chaperone genes such as dnaK in Streptomycetes spp. This analysis was refined in a subsequent work by the same group, by implementation of a high-density microarray Bucca et al.
From this analysis, it emerged that, in S. Moreover, sequence alignment of novel HspR-binding sites highlighted HAIR-like sequences as well as conserved nucleotides extending outside the previously proposed consensus binding sequence.
However, parallel macroarray hybridization of cDNA probes deriving from H. In the last 15 years, the identification of members of the HspR regulon was also pursued through array-based whole transcriptome analyses in several other unrelated bacterial species including Campylobacter jejuni, M.
Overall, these studies suggested that HspR directly represses, alone or in combination with other transcriptional regulators, the transcription of a limited set of genes that encode the major cellular chaperones and heat-shock proteases.
However, indirect effects of hspR disruption affecting transcript abundance of genes involved in diverse cellular processes not strictly related to heat-shock have been observed. For example, in H. Another interesting example of HspR-mediated interplay between the heat-shock response and an unrelated central cellular process was observed in Bifidobacterium breve UCC Zomer et al.
In this microorganism, an SOS response is induced by oxidative stress as well as by heat-shock. Repression of DNA damage repair genes is mediated by the LexA regulator, whose self-cleavage and consequent activation is promoted by the RecA protein.
Induction of RecA expression is probably under the control of HspR under heat-shock conditions, an observation that directly links the repressor to a process employed by Bifidobacterium to respond to an excess of DNA damage Zomer and van Sinderen The characterization of B.
Both CtsR homologs and target sequences were found upstream of clp and other heat-shock genes of several Gram-positive bacteria, an observation that suggested CtsR as a widespread transcriptional repressor involved in heat-shock regulation.
In this respect, it was subsequently shown that, in L. Specifically, it was demonstrated that L. Considering that the positions of the binding sites characterized so far on several promoters of various bacterial species overlap the core promoter region, CtsR should exert its repressive function by sterically occluding RNA polymerase binding to DNA Fig.
The first indications that the prototypical B. Some crucial residues, located within the HTH domain and involved in DNA binding, were identified and found to be highly conserved in the CtsR protein family Fuhrmann et al. The CtsR repressor is historically considered the master regulator of the cellular protein quality control genes of low-GC Gram-positive bacteria.
For example, in the model organism B. In some cases, however, CtsR is also able to regulate the expression of HrcA-dependent genes.
These regulators contact adjacent binding sites and act synergistically to maintain low basal levels of expression of both operons in the absence of stress Chastanet, Fert and Msadek This lactic acid bacterium lacks hrcA in its genome and CtsR has taken over the regulation of typical HrcA targets Grandvalet et al.
Interestingly, some studies have revealed new non-canonical genes regulated by CtsR. These include genes encoding small heat-shock proteins, such as Hsp18 in O. The CtsR master regulator binds the promoters of target genes and represses their transcription under physiological growth conditions. Intriguingly, upon heat-shock, CtsR loses DNA-binding activity and the non-functional protein undergoes regulated proteolytic degradation mediated by the ClpCP protease Elsholz et al.
The first indications that CtsR is a specific target of ClpPC under stress conditions came from in vivo stability assays performed in B. It also became clear that the products of two genes belonging to the CtsR operon, McsA and McsB, play a crucial role in the proteolytic turnover of the repressor.
In particular, mcsB encodes a heat-activated kinase that targets nonfunctional heat-inactivated CtsR, while mcsA codes for an activator of McsB Kirstein et al. The current model for CtsR proteolytic degradation, represented in Fig. Upon heat exposure, McsB is titrated away from ClpC and becomes activated by temperature-dependent autophosphorylation and by interaction with McsA activator. Following this sophisticated dual activation step, McsB acts as an adaptor, targeting non-functional CtsR species and efficiently driving them to degradation.
Active McsB adaptor is downregulated by either dephosphorylation by the cognate phosphatase YwlE or by rapid degradation of the phosphorylated form Elsholz et al. RheA represents an example of heat-shock transcriptional repressor with a restricted distribution among bacteria, being found only in S. The first indications that S. This open reading frame, named orfY and coding for a amino acid polypeptide, is transcribed from a vegetative promoter in the opposite orientation to hsp Moreover, disruption of orfY led to the accumulation of hsp18 transcript, even at low temperature, suggesting a direct or indirect involvement of OrfY in the transcriptional regulation of hsp18 Servant and Mazodier Additional biochemical and genetic studies provided evidence that OrfY was the direct repressor of hsp Thereafter, OrfY was named RheA for r epressor of h eat-shock protein e ighteen.
In particular, expression of S. Considering that the transcription start sites of the divergent hsp18 and rheA genes are separated only by 25 nucleotides and that the —10 boxes of the two promoters partially overlap, it was not surprising to discover that RheA also negatively autoregulates its own transcription Servant, Rapoport and Mazodier RheA represents the first described transcriptional repressor of small heat-shock proteins, anticipating the similar scenario confirmed in O.
Soon after the identification and characterization of the HrcA repressor as a negative regulator of class I heat-shock genes in B. In particular, depletion of the intracellular level of GroESL was associated with high expression of the HrcA-controlled dnaK operon at all temperatures. By contrast, GroESL overexpression led to the hyper-repression of transcription from the target promoter.
It is worth noting that the shifted fragment migrated to the same position in the absence or presence of GroEL, indicating a role for the chaperonin in the modulation of HrcA DNA-binding activity rather than functioning as a co-repressor Mogk et al. Following the formal demonstration of the direct interaction between HrcA and GroE in vitro Reischl, Wiegert and Schumann , a model was proposed for their functional interplay Fig.
Upon stress insult, the accumulation of misfolded polypeptides in the cell sequesters and titrates away the chaperonin, which can no longer interact with the HrcA repressor. Without the presence of bound chaperonin, HrcA loses DNA-binding affinity and repression of class I heat-shock genes is relieved. This model is also supported by the finding that addition of ethanol, treatment with puromycin that causes accumulation of truncated polypeptides in the cytoplasm and overexpression of substrates of GroESL derepress the HrcA regulon Mogk et al.
The same feedback mechanism operates in C. It is worth noting that C. These genes are expressed constitutively throughout the developmental cycle of the bacterium Karunakaran et al. In this system, the characteristic additional C-terminal tail of chlamydial HrcA interfered with repressor binding to the CIRCE element in vitro and also with HrcA-mediated transcriptional repression in vitro and in vivo Chen, Wilson and Tan Intriguingly, the negative effect exerted by the HrcA C-terminal inhibitory region could be counteracted by GroE.
Specifically, it was shown that recombinant GroEL was able to enhance HrcA activity HrcA-binding activity to CIRCE elements in vitro as well as HrcA-mediated repression in vivo and that this effect was more pronounced on the full-length HrcA rather than on the truncated version without the C-terminal tail of the repressor.
Intriguingly, the positive effect of recombinant GroEL on HrcA-binding activity to its operator was shown to be ATP independent, suggesting a non-canonical mechanism of action of the chaperonin Chen, Wilson and Tan The characterization of the GroESL-mediated feedback control of HrcA repressor activity in several distant bacterial species, like for example in C.
The hypothesis that homeostatic control of transcriptional regulators by chaperones might represent a novel common theme in heat-shock gene regulation is confirmed by the characterization of the feedback control of the heat-shock master repressor HspR by DnaK.
Thus, S. This role of DnaK in heat-shock gene regulation was further confirmed both in vitro and in vivo. In particular, it was shown that DnaK was able to stimulate HspR-mediated repression in in vitro transcription assays Bucca et al. Moreover, in vivo depletion of cellular DnaK led to high-level expression of HspR-repressed promoters at normal growth temperature Bucca et al. That is, under normal environmental conditions DnaK interacts with HspR and the ternary complex binds to target DNA, resulting in tight transcriptional repression.
During heat-shock, DnaK is sequestered by unfolded proteins that accumulate in the cytoplasm and HspR is less efficiently able to repress the transcription of target genes whose expression results induced. More recently, a similar DnaK-mediated feedback loop controlling HspR activity has been investigated and characterized in M.
In the context of feedback regulation of heat-shock repressors, H. As described above, H. In analogy with the homologous protein of E. In order to characterize a possible post-transcriptional control over HspR by the DnaK chaperone system, surprisingly it was found that, upon direct protein—protein interaction, CbpA alone was able to negatively modulate HspR binding to target promoters in vitro without contacting the DNA, but only when the repressor was not bound to its operators.
In addition, overexpression of CbpA led to deregulation of heat-shock response in vivo Roncarati, Danielli and Scarlato These findings suggest important considerations and add new perspectives in heat-shock gene regulation. Second, considering the putative functions of CbpA as both a co-chaperone and a nucleoid-associated protein preliminary results suggest that CbpA of H.
Roncarati, personal communication , it would be informative to characterize the functional interplay between HspR and CbpA. This would be a novel example in which heat-shock gene regulation intersects with distinct cellular functions, as for example the maintenance and regulation of the bacterial nucleoid. Two dedicated transcriptional repressors, HrcA and HspR, directly repress three multicistronic operons containing the major chaperone genes of H.
Specifically, the cbp operon encodes a homolog of the E. The hypothetical negative effect of HspR on CbpA activity is indicated by a grey dashed hammerhead marked with a question mark. Experimental data available so far allow to highlight similarities as well as differences among the major heat-shock transcriptional repressors described in the previous sections.
Starting from the mechanism of regulation, they all repress transcription, under normal growth conditions, by sterically occluding RNA polymerase binding to target promoters. In almost all cases, in fact, these repressors interact with the target promoters by binding conserved sequences that overlap core promoter elements, such as —10 and —35 boxes. A closer inspection of their DNA-binding architecture reveals some differences. For instance, B.
Another common feature shared by all heat-shock repressors is their negative autoregulation. This network motif has been suggested to increase the homeostasis of the autoregulated gene product in the context of stochastic gene expression noise, to speed up the response kinetics upon signal perception and to provide more linear dose responses Becskei and Serrano ; Rosenfeld, Elowitz and Alon ; Nevozhay et al.
As shown in Fig. In this respect, Jastrab et al. Specifically, they showed that the heat-shock repressor is actively degraded by the prokaryotic proteasome encoded by this human pathogen, and that degradation is enhanced by an ATP-independent proteasome activator, called PafE, and by HspR denaturation.
The transcriptional regulation of heat-shock genes displays varying degrees of complexity among various microorganisms, reflecting the extreme diversity of genetic regulatory mechanisms in bacteria.
In such simple cases, all the thermoresponsive genes are under the control of a single regulator. For example, in Pseudomonas aeruginosa and in Vibrio cholerae , transcriptional regulation of heat-shock genes is governed by an alternative sigma factor specific for heat stress, similar in sequence and function to E.
As described above, the model organism E. Combinations of heat-shock transcriptional regulators and complex regulatory circuits. A While in some bacterial species heat-shock transcription is controlled exclusively by positive left panel or negative right panel mechanisms of regulation, in several cases positive and negative control strategies coexist central panel.
Black arrows indicate positive regulation, while black hammerheads show negative regulation. B Examples in which distinct transcriptional repressors are employed to control heat-shock gene expression. C Schematic representation of the incoherent feed-forward loop governing heat-shock gene regulation in S. A master regulator, shown in red HspR for H. Among these genes, the groESL operon is also repressed by the HrcA regulator, which is, in turn, under the control of the master repressor.
In other bacterial species, the coexistence of positive and negative control mechanisms, together regulating the expression of distinct sets of heat-shock genes, has been observed. For example, in the Gram-positive pathogen L. A similar situation, in which positive and negative mechanisms are both employed to control separate sets of genes, has been described in other bacteria such as B.
Intriguingly, in cyanobacteria, both positive and negative strategies of transcriptional regulation combine to control expression of some heat-shock genes reviewed by Rajaram, Chaurasia and Apte Furthermore, the histidine kinase Hik34 is involved in the negative regulation of the chaperonin gene Slabas et al. In addition to transcriptional mechanisms, RNA-based regulation of heat-shock genes has also been reported in cyanobacteria.
Expression of the hsp17 gene, coding for a small heat-shock protein also known as HspA, is transcriptionally dependent on the alternative sigma factors SigE and SigB Singh et al. The regulatory schemes found in bacteria that adopt only negative mechanisms of transcriptional regulation could be very simple. However, combinations of repressors and overlaps of their regulons are not rare and sometimes lead to complex regulatory networks.
One of the simplest regulatory scheme is employed, for example, by the human pathogen M. Similarly, a single transcriptional repressor, CtsR, controls heat-shock response in the lactic acid bacterium O. A common manner of combining negative regulators is based on the exploitation of several transcriptional repressors, each specifically controlling a particular set of genes.
An extreme example is represented by S. However, in several other bacterial species, the regulons of two different heat-shock repressors partially overlap and this results in some genes being simultaneously controlled by more than one regulatory protein, rendering these systems more complicated than the examples described above.
HrcA is most commonly involved in those situations, partnering with CtsR in some bacteria, while in some other cases it interacts directly with HspR. Also, there is an upstream CtsR-binding site overlapping the —10 and —35 hexamers Chastanet et al. The two binding sites are in close proximity, separated by only 16 bp.
This tandem operator arrangement, involving a crucial region for transcription initiation, results in the S. This non-redundant dual repression mechanism, also employed very similarly by S. Also, in S. In particular, S. As in S. Strikingly, an almost identical regulatory network has been described in the distantly related Gram-negative human pathogen H. Hence, similarly as for S. HspR instead of CtsR. A detailed analysis of in vitro DNA binding by HrcA and HspR revealed that their operators are arranged in tandem, consistent with the typical architecture for dually regulated promoters, but that the position of the HspR operator maps to far upstream from the core promoter to an atypical position for a repressor Roncarati et al.
Moreover, even though both repressors are required for regulation a single hspR or hrcA mutation leads to complete derepression of the dually controlled promoters , binding in vitro to their respective adjacent operators occurs in an independent, non-cooperative manner. An interesting explanation for the peculiar interaction of heat-shock repressors found in H.
Moreover, considering that all three regulatory interactions are repressive, these circuits seem to represent rare examples of incoherent type-2 feed forward loops Alon ; Danielli, Amore and Scarlato This peculiar network motif is employed to modulate the dynamic behavior of the circuit, greatly speeding up the transcriptional response of target genes upon input stress signals.
Some experimental observations of H. In fact, transcription of groESL was rapidly induced 2 min upon heat-shock in a wild-type genetic background, while in a mutant strain in which the binding site of HspR on the groESL promoter was deleted and, so, the direct connection between HspR and groESL was interrupted , the derepression of groESL transcription was observed only 60 min after temperature challenge Spohn et al.
Even though further experimental characterizations are needed to dissect the dynamic properties of regulatory circuits such as those described above, they could reflect specific evolutionary adaptations to particular needs encountered by bacteria in their specific niches.
Even though, in many cases, clear experimental evidences are still missing, some speculations can be put forward. For instance, the presence in the same organism of diverse regulatory mechanisms involved in the control of distinct as well as overlapping sets of genes might have evolved to allow bacteria a proper response to distinct stresses. The latter aspect has been observed in S. Specifically, two different regulatory patterns have been observed, consisting of constitutive synthesis of GroEL and Hsp18 regulated by HrcA and RheA, respectively at high temperature and transient heat-shock-induced synthesis of HspR-regulated ClpB and DnaK proteins Mazodier et al.
A heat-shock intensity-dependent response differential response of some regulatory systems or individual genes has been documented in different bacterial species, including M. The use of diverse regulatory systems by the same organism to control different heat-shock genes becomes particularly relevant and interesting when it involves the differential regulation of multiple copies of genes coding for the GroEL chaperonins.
It has been observed that a significant proportion of bacterial genomes contains two or more chaperonin genes and, in several instances, these genes appear to be differentially regulated Lund The current notion is that, where present, these multiple chaperonins encoded by duplicated groE genes have evolved a degree of subfunctionalization and the differential regulation observed may reflect the various contexts in which they are needed or the ecological niche they have to cope with. For example, in Bradyrhizobium japonicum , a nitrogen-fixing and roots-nodulating bacterium belonging to the Alphaproteobacteria group, the seven chaperonin genes present in its genome show complex pattern of transcriptional regulation, not limited to heat-shock response.
Genetic analyses provide additional evidences for a link between the regulation of some chaperonins, root nodulation and nitrogen fixation, even though the specificity of chaperonin novel functions appeared not absolute Lund The ability of bacteria to rapidly respond to sudden temperature increase depends on heat-sensing mechanisms that integrate environmental cues to activate appropriate response pathways. To date, various mechanisms of thermoregulation have been described in bacteria and they involve nearly all classes of biomolecules including lipids, proteins and nucleic acids i.
All of these classes can act as thermosensors that detect changes in the environmental temperature and initiate relevant cellular responses. Heat-sensing mechanisms can be direct, by which the temperature directly affects the activity of the sensing biomolecule, or can be indirect, by which the consequences of a sudden temperature increase for example, the accumulation of misfolded proteins in the cytoplasm are detected. Even though temperature is a ubiquitous signal that influences several cellular pathways, this chapter focuses mainly on sensing mechanisms that trigger heat-shock gene expression, and just a few examples of thermosensors involved in the regulation of virulence determinants will be described extensively reviewed by Klinkert and Narberhaus ; Shapiro and Cowen The most rapid way of changing gene expression in response to temperature variations is based on a cis -regulatory element that is part of the mRNA encoding the heat-shock protein to be regulated.
This mechanism of thermoregulation guarantees a very fast response upon signal perception because temperature affects the translation efficiency of both the intracellular pool of mRNA molecules as well as transcription already in progress.
The general principle is based on the formation of zipper-like, temperature-sensitive secondary structures that characterize the mRNAs subjected to this kind of regulation Fig. When such sequence elements are involved in a secondary structure, the recognition and binding of the transcript by the ribosome is hampered, thereby negatively affecting translation of the mRNA. Upon a temperature increase, the secondary structure goes through a rearrangement or partial melting.
Several temperature-sensing RNA sequences, also known as RNA thermometers, have been discovered and characterized in some details in the last two decades and were recently reviewed in a comprehensive and detailed article by Kortmann and Narberhaus The first RNA thermometer was discovered in E.
However, much simpler RNA thermometers exist, as exemplified by the one controlling the expression of the Bradyrhizobium japonicum hspA gene. This cis -regulatory sequence constitutes the founding member of the most abundant class of RNA thermometers called ROSE for r epression o f heat- s hock genes e xpression. ROSE elements are typically involved in the regulation of small heat-shock proteins and they have a length ranging from 60 to about nucleotides.
Initially, it was speculated that they act at the DNA level and their regulatory mechanism was thought to be dependent on the binding of a repressor protein Narberhaus et al.
Noting that it was possible to predict a similar secondary structure for all 15 known ROSE elements, a post-transcriptional mechanism was proposed and verified by a detailed mutational analysis of a ROSE -hspA-lacZ translational fusion Nocker et al. Subsequent extensive molecular and structural characterizations revealed some key features of such kinds of RNA thermometers Chowdhury et al.
Interestingly, it was shown that ROSE-based RNA thermometers comprise two to four stem loops and that they can gradually respond to temperature variation and provide differential levels of expression regulation according to the severity of heat stress. Another class of RNA thermometers is based on a short stretch of four conserved uridines that base pair with the AGGA nucleotide sequence constituting the Shine-Dalgarno sequence.
This element, called fourU, was initially characterized in Salmonella enterica , where it controls the temperature-dependent expression of the agsA gene that encodes for a small heat-shock protein Waldminghaus et al. It is worth noting that thermoregulation based on RNA-sensing structures has not evolved exclusively with respect to heat-shock genes. Mechanisms of heat sensing. During normal growth conditions, a secondary structure forms and sequence elements crucial for efficient initiation of translation are masked and poorly accessible for ribosome binding.
Upon temperature increase, a structural rearrangement or resolution of the structure exposes such sequence elements and translation is enhanced. The DNA region flanked by the H-NS binding sites assumes a curvature at low temperature that allows contacts between H-NS dimers bound to separate binding sites and the formation of a repressive nucleoprotein complex. At higher temperature, this curvature weakens and the promoter is more accessible to RNA polymerase for virF gene transcription.
C Transcriptional regulators as intrinsic heat sensors. Intrinsic heat-sensing repressors are competent for DNA binding at permissive temperature green oval , and transcription of heat-shock genes is repressed. Upon heat challenge, a temperature-induced structural transition shown by a green rectangle lead to a decrease of repressor DNA-binding activity and transcription is derepressed.
D Indirect heat-sensing mechanism mediated by chaperones. The DNA-binding activity of transcriptional regulators is modulated by chaperones.
During normal growth conditions, the chaperone interacts with the heat-shock regulator and exerts its regulatory function. Several positively modulated repressors such as HrcA and HspR in some bacterial species gain DNA-binding activity upon chaperone interaction, while in other instances the interaction with the chaperone results in the sequestration of the activator for example, E. Following heat stress, chaperones are sequestered by misfolded proteins that accumulate in the cytoplasm and transcriptional regulators are released and their DNA-binding activity results are positively or negatively affected.
In some cases, temperature variations can be directly sensed by the DNA of the bacterial cell. Several physiological metabolic pathways of a microorganism are influenced by the external conditions experienced in its ecological niche. For this reason, external fluctuating conditions that affect metabolic processes, including osmotic and heat-shock, can ultimately influence the global level of DNA supercoiling Hsieh, Burger and Drlica ; Dorman and Corcoran Considering that DNA supercoiling can influence gene transcription, DNA can be considered as a thermosensor of environmental temperature change acting through variations of the global topological state of the chromosome in response to external stimuli.
One of the primary parameters of DNA topology that responds to temperature changes is plasmid supercoiling. In some other cases, however, local DNA structures mediate temperature sensing and affect transcription of neighboring genes.
Some DNA sequences identified in E. When these DNA regions are proximal to promoters, the local conformational variation induced by heat-shock is transduced into a modulation of RNA polymerase binding efficiency, thereby affecting the transcription of the downstream genes Nickerson and Achberger An example is represented by the region upstream of the plc gene, encoding the phospholipase C PLC in Clostridium perfrigens Katayama et al.
By using in vitro transcription assays, it was demonstrated that the stimulatory effect on the promoter activity of the A-tract sequence was temperature dependent, probably due to changes in the bending angle upon temperature fluctuations. Besides the direct activation of transcription mediated by bent DNA through the facilitation of RNA polymerase binding, temperature-dependent local DNA curvatures can indirectly regulate the efficiency of transcription by affecting the interaction of proteins with a regulatory role on gene expression.
One of the best studied examples concerns the temperature-dependent regulation of virF transcription in Shigella flexneri , a pathogenic bacterium able to invade human intestinal epithelium Fig. The AraC-like VirF regulator, a protein that triggers the activation of several genes with invasive functions, must be expressed only after the shift from the outside environment to the host.
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Song Huang , Song Huang. Oxford Academic. Xiao Dong Chen. Editor: Abelardo Margolles. Revision received:. Select Format Select format. Permissions Icon Permissions. Abstract The heat resistance of lactic acid bacteria LAB has been extensively investigated due to its highly practical significance.
Open in new tab Download slide. Cells in maltose solutions with CaCl 2. Cells in lactose solutions with CaAc. Open in new tab. A defined range of guard cell calcium oscillation parameters encodes stomatal movements.
Google Scholar Crossref. Search ADS. Significance of inoculum size in the lag time of Listeria monocytogenes. Effect of Lactobacillus plantarum P-8 on lipid metabolism in hyperlipidemic rat model. Effects of pH, calcium-complexing agents and milk solids concentration on formation of soluble protein aggregates in heated reconstituted skim milk.
Micro-organism inactivation during drying of small droplets or thin-layer slabs — a critical review of existing kinetics models and an appraisal of the drying rate dependent model. Environmental adaptation of probiotic lactobacilli towards improvement of performance during spray drying. Calcium oscillations increase the efficiency and specificity of gene expression.
Espeche Turbay. Release of the cell-envelope-associated proteinase of Lactobacillus delbrueckii subspecies lactis CRL is dependent upon pH and temperature. Role of calcium in activity and stability of the lactococcus lactis cell envelope proteinase. Google Scholar PubMed. Joint working group report on drafting. It took around two years to reach The researchers propagated a control population, also descended from the common ancestor, at 37 degrees C.
Exhaustive analysis of cell physiology, protein expression, and genome sequences revealed a number of significant changes. Compared with the control group, the adapted lines of E. This was accompanied by reduced growth rates, showing that survival came at a cost in terms of overall fitness, a typical indicator of genomic mutations.
One of the most striking changes measured was a fold increase in GroE levels, more than five times what a normal heat shock response in E. Further analyses of heat shock genes and proteins ruled out other mechanisms -- beyond the role of GroE alone -- as being critical for evolution for life at GroE chaperones are known to play an active role in assisting the folding process of other proteins, especially in cases where mutations that could cause improper folding threaten the survival of the cell.
This experiment shows that they likely play a uniquely important role -- by mitigating the potentially damaging effects of accumulating mutations on protein folding -- in the evolution of heat resistance in E. Beyond yielding insights into evolutionary history, Winter says, further research on these highly conserved mechanisms could shed light on how organisms evolve in response to climate-related stresses in the future. Materials provided by Technische Universitaet Muenchen. Note: Content may be edited for style and length.
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