ClpS is an essential component of the N-end rule pathway in Escherichia coli.

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.

Protein folding and degradation in bacteria: to degrade or not to degrade? That is the question.

In Escherichia coli protein quality control is carried out by a protein network, comprising chaperones and proteases. Central to this network are two protein families, the AAA+ and the Hsp70 family. The major Hsp70 chaperone. DnaK, efficiently prevents protein aggregation and supports the refolding of damaged proteins. In a special case, DnaK, together with the assistance of the AAA+ protein ClpB, can also refold aggregated proteins. Other Hsp70 systems have more specialized functions in the cell, for instance HscA appears to be involved in the assembly of Fe/S proteins. In contrast to ClpB, many AAA+ proteins associate with a peptidase to form proteolytic machines which remove irreversibly damaged proteins from the cellular pool. The AAA+ component of these proteolytic machines drives protein degradation. They are required not only for recognition of the substrate but also for substrate unfolding and translocation into the proteolytic chamber. In many cases, specific adaptor proteins modify the substrate binding properties of AAA+ proteins. While chaperones and proteases do not appear to directly cooperate with each other, both systems appear to be necessary for proper functioning of the cell and can, at least in part, substitute for one another.

Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol.

We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In DeltarpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5-10% and 20-30% of total protein aggregated at 30 degrees C and 42 degrees C respectively. The aggregates contained 350-400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30 degrees C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low-level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to DeltarpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30 degrees C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK-depleted cells. At 42 degrees C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42 degrees C.

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.

UNLABELLED: We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (

Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network.

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid binding, and of the disaggregation/refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.

Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

Post-transcriptional regulation of the Bacillus subtilis dnaK operon.

The heptacistronic dnaK heat shock operon of Bacillus subtilis consists of the genes hrcA, grpE, dnaK, dnaJ, orf35, orf28 and orf50. It is controlled by the CIRCE/HrcA operator/repressor system and specifies three primary transcripts, two of which are processed into three different products. We have analysed the regulatory consequences of this complex transcriptional organization in detail. First, the seven genes were heat induced to different extents at the mRNA level and can be classified into three groups by their induction factors. This differential induction was also reflected at the protein level. Secondly, the cellular amounts of the proteins HrcA, DnaK and DnaJ in B. subtilis differed drastically both under non-heat shock conditions and after thermal upshock. Thirdly, Northern blot analyses demonstrated that an mRNA-processing reaction generating products of differential stabilities plays an essential role during the regulation of gene expression. A crucial factor determining the low stability of two transcripts is the presence of the CIRCE element at their 5' ends. We demonstrate that CIRCE leads to the destabilization of mRNAs, but only if it is located in the immediate vicinity of a Shine-Dalgarno sequence. These results show that B. subtilis is using various, especially post-transcriptional, regulatory mechanisms to fine tune the expression of the individual genes of the heptacistronic dnaK operon.

Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones.

Hsp70 chaperones assist a large variety of protein folding processes within the entire lifespan of proteins. Central to these activities is the regulation of Hsp70 by DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP, a key step that closes its substrate-binding cavity and thus allows stable binding of substrate. We show that DnaJ stimulates ATP hydrolysis by Escherichia coli Hsp70, DnaK, very efficiently to >1000-fold, but only if present at high (micromolar) concentration. In contrast, the chaperone activity of DnaK in luciferase refolding was maximal at several hundredfold lower concentration of DnaJ. However, DnaJ was capable of maximally stimulating the DnaK ATPase even at this low concentration, provided that protein substrate was present, indicating synergistic action of DnaJ and substrate. Peptide substrates were poorly effective in this synergistic action. DnaJ action required binding of protein substrates to the central hydrophobic pocket of the substrate-binding cavity of DnaK, as evidenced by the reduced ability of DnaJ to stimulate ATP hydrolysis by a DnaK mutant with defects in substrate binding. At high concentrations, DnaJ itself served as substrate for DnaK in a process considered to be unphysiological. Mutant analysis furthermore revealed that DnaJ-mediated stimulation of ATP hydrolysis requires communication between the ATPase and substrate-binding domains of DnaK. This mechanism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrate binding and to avoid jamming of the DnaK chaperone with peptides. It probably is conserved among Hsp70 family members and is proposed to account for their functional diversity.

Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

The Bacillus subtilis htpG gene is not involved in thermal stress management.

To study the influence of the htpG gene on thermal stress management in Bacillus subtilis, two different kinds of htpG mutation were constructed. In one case, the gene was inactivated by insertion of a cat cassette in to the coding region; htpG was thus found to be non-essential. In the second case, the htpG gene was fused to a xylose-dependent promoter, allowing expression of the gene to be controlled. In the absence of HtpG protein, recovery of cells from a heat shock at 53 degrees C was retarded, and this delay could be eliminated by overproduction of HtpG. While htpG is not involved in the development of induced thermotolerance, DnaK and GroE proteins are absolutely required. Overproduction of class I heat-shock proteins prior to shifting cells to a lethal temperature is important but not sufficient for the development of intrinsic thermotolerance. It could be shown that the HtpG protein does not act as a cellular thermometer in B. subtilis.

Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes.

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.

Nonnative proteins induce expression of the Bacillus subtilis CIRCE regulon.

The chaperone-encoding groESL and dnaK operons constitute the CIRCE regulon of Bacillus subtilis. Both operons are under negative control of the repressor protein HrcA, which interacts with the CIRCE operator and whose activity is modulated by the GroESL chaperone machine. In this report, we demonstrate that induction of the CIRCE regulon can also be accomplished by ethanol stress and puromycin. Introduction of the hrcA gene and a transcriptional fusion under the control of the CIRCE operator into Escherichia coli allowed induction of this fusion by heat shock, ethanol stress, and overproduction of GroESL substrates. The expression level of this hrcA-bgaB fusion inversely correlated with the amount of GroE machinery present in the cells. Therefore, all inducing conditions seem to lead to induction via titration of the GroE chaperonins by the increased level of nonnative proteins formed. Puromycin treatment failed to induce the sigmaB-dependent general stress regulon, indicating that nonnative proteins in general do not trigger this response. Reconstitution of HrcA-dependent heat shock regulation of B. subtilis in E. coli and complementation of E. coli groESL mutants by B. subtilis groESL indicate that the GroE chaperonin systems of the two bacterial species are functionally exchangeable.

The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis.

Class I heat-inducible genes in Bacillus subtilis consist of the heptacistronic dnaK and the bicistronic groE operon and form the CIRCE regulon. Both operons are negatively regulated at the level of transcription by the HrcA repressor interacting with its operator, the CIRCE element. Here, we demonstrate that the DnaK chaperone machine is not involved in the regulation of HrcA and that the GroE chaperonin exerts a negative effect in the post-transcriptional control of HrcA. When expression of the groE operon was turned off, the dnaK operon was significantly activated and large amounts of apparently inactive HrcA repressor were produced. Overproduction of GroEL, on the other hand, resulted in decreased expression of the dnaK operon. Introduction of the hrcA gene and its operator into Escherichia coli was sufficient to elicit a transient heat shock response, indicating that no additional Bacillus-specific gene(s) was needed. As in B. subtilis, the groEL gene of E. coli negatively influenced the activity of HrcA. HrcA could be overproduced in E. coli, but formed inclusion bodies which could be dissolved in 8 M urea. Upon removal of urea, HrcA had a strong tendency to aggregate, but aggregation could be suppressed significantly by the addition of GroEL. Purified HrcA repressor was able specifically to retard a DNA fragment containing the CIRCE element, and the amount of retarded DNA was increased significantly in the presence of GroEL. These results suggest that the GroE chaperonin machine modulates the activity of the HrcA repressor and therefore point to a novel function of GroE as a modulator of the heat shock response.

Cloning and sequencing of the hrcA gene of Bacillus stearothermophilus.

We report on cloning and sequencing of a 2.0-kb PCR fragment of chromosomal DNA from thermophilic Bacillus stearothermophilus carrying the complete hrcA gene. In addition, this amplicon contains the 3' end of an open reading frame exhibiting significant homology to the hemN gene of Bacillus subtilis (Bs) and other bacterial species. The hrcA gene could complement an Bs hrcA deletion mutant by repressing expression of class I heat shock (HS) genes. Furthermore, we could show that the HrcA protein derived from the thermophilic microorganism responds to HS in a similar way as reported for the Bs HrcA protein.

Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable beta-galactosidase.

Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable beta-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3'- and 5'-end of the amyE gene (encoding alpha-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.

The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion.

The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP- and Zn(2+)-dependent protease anchored in the cytoplasmic membrane via two N-terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat-cassette near the 5' end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat-shock response. The induction profiles of representative genes of the CIRCE and sigma-B regulon and class III heat-shock genes ion and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The alpha-amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of alpha-amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint.

The dnaK operon of Bacillus subtilis is heptacistronic.

In 1992, we described the cloning and sequencing of the dnaK locus of Bacillus subtilis which, together with transcriptional studies, implied a tetracistronic structure of the operon consisting of the genes hrcA, grpE, dnaK, and dnaJ. We have repeated the Northern blot analysis, this time using riboprobes instead of oligonucleotides, and have detected a heat-inducible 8-kb transcript, suggesting the existence of additional heat shock genes downstream of dnaJ. Cloning and sequencing of that region revealed the existence of three novel heat shock genes named orf35, orf28, and orf50, extending the tetra- into a heptacistronic operon. This is now the largest dnaK operon to be described to date. The three new genes are transcribed as a part of the entire dnaK operon (8.0-kb heptacistronic heat-inducible transcript) and as part of a suboperon starting at an internal vegetative promoter immediately upstream of dnaJ (4.3-kb tetracistronic non-heat-inducible transcript). In addition, the Northern blot analysis detected several processing products of these two primary transcripts. To demonstrate the existence of the internal promoter, a DNA fragment containing this putative promoter structure was inserted upstream of a promoterless bgaB gene, resulting in the synthesis of beta-galactosidase. Challenging this transcriptional fusion with various stress factors did not result in the activation of this promoter. To assign a biological function to the three novel genes, they have each been inactivated by the insertion of a cat cassette. All of the mutants were viable, and furthermore, these genes are (i) not essential for growth at high temperatures, (ii) not involved in the regulation of the heat shock response, and (iii) sporulation proficient. Blocking transcription of the suboperon from the upstream heat-inducible promoter did not impair growth and viability at high temperatures.

Integrative vectors for constructing single-copy transcriptional fusions between Bacillus subtilis promoters and various reporter genes encoding heat-stable enzymes.

Here, we report on the construction of three integrative plasmids for Bacillus subtilis (Bs) allowing in vitro construction of transcriptional fusions. These plasmids contain a neomycin- or tetracycline-resistance cassette and one of three promoterless genes: bgaB (encoding beta-galactosidase), cat (chloramphenicol acetyltransferase), or xylE (catechol 2,3-dioxygenase). All cassettes are flanked by the 3'- and 5'-ends of the amyE gene (encoding alpha-amylase) allowing integration of these cassettes at the amyE locus of the Bs chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding bla gene. Four unique restriction sites can be used for insertion of restriction fragments carrying promoter fragments. All three reporter genes express heat-stable enzymes (stable up to at least 50 degrees C for 30 min) as shown here. We would like to point to the modular nature of these plasmids where the three reporter genes and the two resistance cassettes can be combined in any permutation. The versatility of the promoter-probe vectors was demonstrated by the integration of the promoters of the dnaK and groE operons of Bs and following their heat-inducible expression.

A xylose-inducible Bacillus subtilis integration vector and its application.

The construction of a xylose-inducible expression vector is described. This vector allows the integration of any gene, coding for its authentic protein, at the amyE locus of Bacillus subtilis (Bs). The controlable expression cassette consists of the repressor-encoding gene and the promoter of the Bacillus megaterium-derived operon for xylose utilization, sandwiched between the 5'- and 3'-ends of amyE. This thereby allows insertion of in vitro constructed transcriptional fusions at the amyE locus of the Bs chromosome. The versatility of this expression system was tested by fusing three different heat-shock genes to the xylose-inducible promoter and following their expression by Western immunoblot analysis. Whereas no increase in the amount of heat-shock protein could be detected under non-inducing conditions when compared to the isogenic wild-type strain, the three proteins were strongly induced after addition of xylose, depending on the gene. To determine the tightness and the induction factor of the system more accurately, the bgaB gene encoding a heat-stable beta-galactosidase (beta Gal) was analyzed. The background activity of beta Gal increased by a factor of at least 200 after addition of xylose. The system is not subject to catabolite, but rather to glucose repression.