Search Results (7)

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which counteract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two repressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42 °C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regulated genes. Furthermore, we show that this impairment of HrcA binding at 42 °C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37 °C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combination with HrcA, the transcription of chaperones’ genes, is unaffected by heat-stress up to 45 °C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein–protein interaction. In conclusion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chaperonin. Full article

Rice plants often encounter high temperature stress, but the associated coping strategies are poorly understood. It is known that a prior shorter exposure to high temperature, called thermo-priming, generally results in better adaptation of the plants to subsequent exposure to high temperature stress. High throughput sequencing of transcript and small RNA libraries of rice seedlings primed with short exposure to high temperature followed by high temperature stress and from plants exposed to high temperature without priming was performed. This identified a number of transcripts and microRNAs (miRs) that are induced or down regulated. Among them osa-miR531b, osa-miR5149, osa-miR168a-5p, osa-miR1846d-5p, osa-miR5077, osa-miR156b-3p, osa-miR167e-3p and their respective targets, coding for heat shock activators and repressors, showed differential expression between primed and non-primed plants. These findings were further validated by qRT-PCR. The results indicate that the miR-regulated heat shock proteins (HSPs)/heat shock transcription factors (HSFs) may serve as important regulatory nodes which are induced during thermo-priming for plant survival and development under high temperatures. Full article

The heat-shock response is defined by the transient gene-expression program that leads to the rapid accumulation of heat-shock proteins. This evolutionary conserved response aims at the preservation of the intracellular environment and represents a crucial pathway during the establishment of host–pathogen interaction. In the food-borne pathogen Campylobacter jejuni two transcriptional repressors, named HspR and HrcA, are involved in the regulation of the major heat-shock genes. However, the molecular mechanism underpinning HspR and HrcA regulatory function has not been defined yet. In the present work, we assayed and mapped the HspR and HrcA interactions on heat-shock promoters by high-resolution DNase I footprintings, defining their regulatory circuit, which governs C. jejuni heat-shock response. We found that, while DNA-binding of HrcA covers a compact region enclosing a single inverted repeat similar to the so-called Controlling Inverted Repeat of Chaperone Expression (CIRCE) sequence, HspR interacts with multiple high- and low-affinity binding sites, which contain HspR Associated Inverted Repeat (HAIR)-like sequences. We also explored the DNA-binding properties of the two repressors competitively on their common targets and observed, for the first time, that HrcA and HspR can directly interact and their binding on co-regulated promoters occurs in a cooperative manner. This mutual cooperative mechanism of DNA binding could explain the synergic repressive effect of HspR and HrcA observed in vivo on co-regulated promoters. Peculiarities of the molecular mechanisms exerted by HspR and HrcA in C. jejuni are compared to the closely related bacterium H. pylori that uses homologues of the two regulators. Full article

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators. Full article

Bacteria respond to different environmental stresses by reprogramming the transcription of specific genes whose proper expression is critical for their survival. In this regard, the heat-shock response, a widespread protective mechanism, triggers a sudden increase in the cellular concentration of different proteins, including molecular chaperones and proteases, to preserve protein folding and maintain cellular homeostasis. In the medically important gastric pathogen Helicobacter pylori the regulation of the principal heat-shock genes is under the transcriptional control of two repressor proteins named HspR and HrcA. To define the HrcA regulon, we carried out whole transcriptome analysis through RNA-sequencing, comparing the transcriptome of the H. pylori G27 wild type strain to that of the isogenic hrcA-knockout strain. Overall, differential gene expression analysis outlined 49 genes to be deregulated upon hrcA gene inactivation. Interestingly, besides controlling the transcription of genes coding for molecular chaperones and stress-related mediators, HrcA is involved in regulating the expression of proteins whose function is linked to several cellular processes crucial for bacterial survival and virulence. These include cell motility, membrane transporters, Lipopolysaccharide modifiers and adhesins. The role of HrcA as a central regulator of H. pylori transcriptome, as well as its interconnections with the HspR regulon are here analyzed and discussed. As the HrcA protein acts as a pleiotropic regulator, influencing the expression of several stress-unrelated genes, it may be considered a promising target for the design of new antimicrobial strategies. Full article

The ability to gauge the surroundings and modulate gene expression accordingly is a crucial feature for the survival bacterial pathogens. In this respect, the heat-shock response, a universally conserved mechanism of protection, allows bacterial cells to adapt rapidly to hostile conditions and to survive during environmental stresses. The important and widespread human pathogen Helicobacter pylori enrolls a collection of highly conserved heat-shock proteins to preserve cellular proteins and to maintain their homeostasis, allowing the pathogen to adapt and survive in the hostile niche of the human stomach. Moreover, various evidences suggest that some chaperones of H. pylori may play also non-canonical roles as, for example, in the interaction with the extracellular environment. In H. pylori, two dedicated transcriptional repressors, named HspR and HrcA, homologues to well-characterized regulators found in many other bacterial species, orchestrate the regulation of heat-shock proteins expression. Following twenty years of intense research, characterized by molecular, as well as genome-wide, approaches, it is nowadays possible to appreciate the complex picture representing the heat-shock regulation in H. pylori. Specifically, the HspR and HrcA repressors combine to control the transcription of target genes in a way that the HrcA regulon results embedded within the HspR regulon. Moreover, an additional level of control of heat-shock genes’ expression is exerted by a posttranscriptional feedback regulatory circuit in which chaperones interact and modulate HspR and HrcA DNA-binding activity. This review recapitulates our understanding of the roles and regulation of the most important heat-shock proteins of H. pylori, which represent a crucial virulence factor for bacterial infection and persistence in the human host. Full article

Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ∆ctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ∆ctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria. Full article