Controlling Inverted Repeat Of Chaperone Expression Research Articles

Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA.

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.

CIRCE element evolved for the coordinated transcriptional regulation of bacterial duplicate groELs

Chaperonin groEL genes are duplicated in approximately 20% of bacteria, and the duplicates are differentially transcribed due to their divergent functions. The coordinated regulation of this differential transcription is as yet undetermined. In this study, we reported that the controlling inverted repeat of chaperone expression (CIRCE) element (the HrcA-binding site located upstream of the promoter) evolved for the transcriptional regulation of duplicate groELs. CIRCE composition and locations were found to be phylogenetically conserved in bacterial taxa. Myxococcus xanthus DK1622 has two CIRCE elements (CIRCE1groESL1 and CIRCE2groESL1) in the promoter region of groESL1 and one CIRCE element (CIRCEgroEL2) before groEL2. We also found that negative HrcA and positive σ32 regulators coordinated the transcription of duplicate groELs, and that the double deletion in DK1622 eliminated transcriptional differences and reduced the heat-shock responses of groELs. In vitro binding assays showed that HrcA protein binding was biased towards CIRCE1groESL1, followed by CIRCEgroEL2, but that HrcA proteins failed to bind with CIRCE2groESL1. Mutation experiments revealed that single-nucleotide mutations in the inverted repeat regions changed the HrcA-binding abilities of CIRCEs. We constructed an in vivo transcription-regulation system in Escherichia coli to pair each of the regulators with a groEL promoter. The results indicated that the transcriptional regulation performed by HrcA and σ32 was biased towards the groEL2 and groEL1 promoters, respectively. Based on promoter-sequence characteristics, we proposed a model of the coordinated regulation of the transcription of duplicate groELs in M. xanthus DK1622.

The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR) Analysis

Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5′ noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

Hydrophobic surface exposure-mediated CIRCE repression by the Ca 2+-sensor, HrcA, in Streptococcus pneumoniae

Heat shock-induced expression of the CIRCE ( controlling inverted repeat of chaperone expression) regulon in Streptococcus pneumoniae was examined in the presence of Ca 2+. In the presence of the Ca 2+, thermo-resistance and expression of the CIRCE regulon of S. pneumoniae were significantly repressed. In addition, hydrophobicity of the HrcA was increased in the presence of the metal ion suggesting that Ca 2+ induces conformational changes such as exposure of the hydrophobic surfaces of HrcA, which facilitate binding to GroEL. This in turn enhances access to CIRCE and leads to repression of the dnaK and groE operons.

Ca2+‐dependent expression of the CIRCE regulon inStreptococcus pneumoniae

DnaK and GroEL play a pivotal role in protein folding, and promote cell proliferation and survival. In Gram-positive and several Gram-negative bacteria, HrcA represses the transcription of dnaK and groE operons by binding to the highly conserved CIRCE (controlling inverted repeat of chaperone expression) operator sequence in the presence of GroEL. HrcA may respond to environmental stress and various other factors that modulate the transcription of the dnaK and groE operons. However, the mechanisms by which these factors modulate the activity of HrcA remain elusive. Here, we show that the thermoresistance of Streptococcus pneumoniae is significantly repressed in the presence of Ca2+. Furthermore, heat shock-induced expression of the CIRCE regulon in S. pneumoniae is repressed in the presence of Ca2+, although to a lesser degree than in the hrcA mutant, strongly suggesting that HrcA inhibits expression of the CIRCE regulon in a Ca2+-dependent manner. Although HrcA does not bind directly to Ca2+, its hydrophobicity is increased in the presence of the metal ion. Taken together, our observations suggest that Ca2+ induces conformational changes, such as exposure of the hydrophobic surfaces of HrcA, which facilitate binding to GroEL. Alternatively, the presence of Ca2+ may facilitate GroEL in interacting freely with HrcA. This, in turn, enhances access to CIRCE and leads to repression of the dnaK and groE operons in S. pneumoniae.

The immunogenicity of Rhodococcus equi GroEL2-based vaccines in a murine model.

Rhodococcus equi is a significant intracellular bacterial pathogen in foals. However, at present there is no commercially available vaccine for the prevention of R. equi-induced disease in these animals. Studies have shown that GroEL based vaccines can afford protection against some intracellular pathogens. In this study, the R. equi gene encoding the heat shock protein GroEL2 was cloned and sequenced, with a view to using it as a vaccine candidate. The promoter region of the gene contained two copies of controlling inverted repeat of chaperone expression (CIRCE) motifs, which are well-recognised transcriptional regulators of bacterial heat shock proteins. The R. equi GroEL2 was expressed in E. coli BL21 DE3 with a C-terminal His-tag and sequenced to confirm its identity. The R. equi purified His-tagged GroEL2 protein and a groEL2-based DNA vaccine were used in separate experiments to immunise BALB/c mice. The recombinant protein-based vaccine elicited a mixed Th1/Th2 response whereas the DNA vaccine was found to elicit a predominantly Th1 biased immune response. However, when vaccinated mice were challenged intravenously with 1.5 x 10(7) R. equi neither vaccine elicited enhanced bacterial clearance from the spleen or liver in this model. The reasons for this apparent lack of success are discussed.

Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex, indicating a role as a thermosensor.

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.

Molecular characterization and regulatory analysis of dnaK operon of halophilic lactic acid bacterium Tetragenococcus halophila

We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region up-stream of hrcA. The T. halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T. halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T. halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3–4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.

Molecular Characterization and Regulatory Analysis of dnaK Operon of Halophilic Lactic Acid Bacterium Tetragenococcus halophila.

We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region up-stream of hrcA. The T. halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T. halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T. halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3–4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.

Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro.

HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified B. thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B. thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B. thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B. thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.

Cloning, sequencing, and transcriptional analysis of the dnaK heat shock operon of Listeria monocytogenes

The complete dnaK operon of Listeria monocytogenes was isolated by chromosome walking using the previously cloned dnaK gene as a probe. Molecular analysis of the locus identified 6 genes in the order hrcA, grpE, dnaK, dnaJ, orf35, and orf29. Primer extension analysis revealed 3 transcription start sites-S1, S2, and S3-upstream of the hrcA, grpE, and dnaJ, respectively. The transcription from S1 was heat inducible. Analysis of the sequences revealed the consensus promoter sequences of gram-positive bacteria, P1 and P2 upstream of the hrcA and dnaJ, respectively. The hrcA gene and a regulatory sequence, designated CIRCE (controlling inverted repeat of chaperone expression), play a role in the regulation of expression of the dnaK locus in response to heat shock in several gram-positive bacteria. Their presence upstream of the dnaK locus in L. monocytogenes suggested a similar regulatory mechanism for the transcription initiated at the promoter, P1. Northern blot analysis led to the detection of 4 mRNA species of 4.9 kb, 3.6 kb, 3.6 kb, and 1.2 kb; the first 2 species were heat inducible. The current results indicate that 4 distinct transcripts directed by 3 promoters are involved in the expression of the dnaK operon of L. monocytogenes.

Sequence analysis and heterologous expression of the groE genes from Thermoanaerobacter sp. Rt8.G4.

The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp. Rt8.G4 (TRt) have been isolated, sequenced and analysed. The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon. A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES. At optimum growth temperature (65 degreesC) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3-10 degreesC. Functionally intact recombinant TRt chaperonins were produced in Escherichia coli. However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host.

Cloning, characterization, and transcriptional analysis of a gene encoding an alpha-crystallin-related, small heat shock protein from the thermophilic cyanobacterium Synechococcus vulcanus.

hspA, a gene encoding a 16-kDa heat-induced protein from the thermophilic cyanobacterium Synechococcus vulcanus, has been cloned and sequenced. The deduced amino acid sequence of the gene product showed significant homology to sequences of the family of alpha-crystallin-related, small heat shock proteins. A monocistronic mRNA of hspA increased transiently in response to heat shock. The heat shock induction occurred at a vegetative promoter but without the CIRCE (controlling inverted repeat of chaperone expression) element.

Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance.

The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.

Thioredoxin is an essential protein induced by multiple stresses in Bacillus subtilis.

Thioredoxin, a small, ubiquitous protein which participates in redox reactions through the reversible oxidation of its active center dithiol to a disulfide, is an essential protein in Bacillus subtilis. A variety of stresses, including heat or salt stress or ethanol treatment, strongly enhanced the synthesis of thioredoxin in B. subtilis. The stress induction of the monocistronic trxA gene encoding thioredoxin occurs at two promoters. The general stress sigma factor, sigmaB, was required for the initiation of transcription at the upstream site, S(B), and the promoter preceding the downstream start site, S(A), was presumably recognized by the vegetative sigma factor, sigmaA. In contrast to the heat-inducible, sigmaA-dependent promoters preceding the chaperone-encoding operons groESL and dnaK, no CIRCE (for controlling inverted repeat of chaperone expression) was present in the vicinity of the start site, S(A). The induction patterns of the promoters differed, with the upstream promoter displaying the typical stress induction of sigmaB-dependent promoters. Transcription initiating at S(A), but not at S(B), was also induced after treatment with hydrogen peroxide or puromycin. Such a double control of stress induction at two different promoters seems to be typical of a subgroup of class III heat shock genes of B. subtilis, like clpC, and it either allows the cells to raise the level of the antioxidant thioredoxin after oxidative stress or allows stressed cells to accumulate thioredoxin. These increased levels of thioredoxin might help stressed B. subtilis cells to maintain the native and reduced state of cellular proteins.

Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK

An inverted repeat sequence known as CIRCE (controlling inverted repeat of chaperone expression) in the Bacillus subtilis groE operon has been suggested to function as an operator. To identify the regulatory gene directly or indirectly involved in CIRCE-mediated heat-inducible groE expression, B. subtilis WBG2, carrying an integrated groE-bgaB transcription fusion in the amyE locus, was mutagenized. Dark blue colonies formed at 37 degrees C represent mutants which constitutively produce BgaB (a thermostable beta-galactosidase) at high levels. Seven mutants (WBG101 to WBG107) were selected for further characterization. They all overproduced BgaB, GroEL, and DnaK simultaneously at 37 degrees C. These mutants could be restored to normal by introducing a plasmid carrying a functional copy of orf39, the first gene in the B. subtilis dnaK operon. Genomic sequencing of these mutants demonstrated that they all carried a single mutation in orf39. These mutations can be divided into three groups: (i) Gly-307 to Asp, (ii) Ser-122 to Phe, and (iii) Gly-63 to Glu. By using a binary vector system in E. coli, production of ORF39 was found to negatively regulate the expression of groE-bgaB in a CIRCE-specific manner. Under the heat shock condition, the negative regulation mediated by ORF39 was abolished. Mobility shift of the CIRCE-containing probe was also observed with the crude extract prepared from the E. coli strain that overproduced ORF39. Therefore, ORF39 is the negative regulatory factor which regulates both groE and dnaK expression in B. subtilis. It is likely to function as a CIRCE-specific repressor.

CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis

The dnaK and groESL operons of Bacillus subtilis are preceded by a potential sigma 43 promoter sequence (recognized by the vegetative sigma factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin.