Clp-mediated proteolysis in Gram-positive bacteria is autoregulated by the stability of a repressor.
ABSTRACT: The heat shock proteins ClpC and ClpP are subunits of an ATP-dependent protease of Bacillus subtilis. Under non-stressed conditions, transcription of the clpC and clpP genes is negatively regulated by CtsR, the global repressor of clp gene expression. Here, CtsR was proven to be a specific substrate of the ClpCP protease under stress conditions. Two proteins of former unknown function, McsA and McsB, which are also encoded by the clpC operon, act as modulators of CtsR repression. McsA containing zinc finger motifs stabilizes CtsR under non-stressed conditions. McsB, a putative kinase, can inactivate CtsR by modification to remove the repressor from the DNA and to target CtsR for degradation by the ClpCP protease during stress. Thus, clp gene expression in Gram-positive bacteria is autoregulated by a novel mechanism of controlled proteolysis, a circuit of down-regulation by stabilization and protection of a transcription repressor, and induction by presenting the repressor to the protease. Thereby, the ClpC ATPase, a member of the Hsp100 family, was identified as a positive regulator of the heat shock response.
Project description:Cells of the soil bacterium Bacillus subtilis have to adapt to fast environmental changes in their natural habitat. Here, we characterized a novel system in which cells respond to heat shock by regulatory proteolysis of a transcriptional repressor CtsR. In B. subtilis, CtsR controls the synthesis of itself, the tyrosine kinase McsB, its activator McsA and the Hsp100/Clp proteins ClpC, ClpE and their cognate peptidase ClpP. The AAA+ protein family members ClpC and ClpE can form an ATP-dependent protease complex with ClpP and are part of the B. subtilis protein quality control system. The regulatory response is mediated by a proteolytic switch, which is formed by these proteins under heat-shock conditions, where the tyrosine kinase McsB acts as a regulated adaptor protein, which in its phosphorylated form activates the Hsp100/Clp protein ClpC and targets the repressor CtsR for degradation by the general protease ClpCP.
Project description:The soil bacterium Bacillus subtilis possesses a fine-tuned and complex heat stress response system. The repressor CtsR, whose activity is regulated by its modulators McsA and McsB, controls the expression of the cellular protein quality control genes clpC, clpE and clpP. Here, we show that the interaction of McsA and McsB with CtsR results in the formation of a ternary complex that not only prevents the binding of CtsR to its target DNA, but also results in a subsequent phosphorylation of McsB, McsA and CtsR. We further demonstrate that McsB is a tyrosine kinase that needs McsA to become activated. ClpC inhibits the kinase activity of McsB, indicating a direct role in initiating CtsR-controlled heat shock response. Interestingly, the kinase domain of McsB is homologous to guanidino phosphotransferase domains originating from eukaryotic arginine and creatine kinases. Mutational analysis of key residues of the guanidino kinase domain demonstrated that McsB utilizes this domain to catalyze the tyrosine phosphorylation. McsB represents therefore a new kind of tyrosine kinase, driven by a guanidino phosphotransferase domain.
Project description:In Bacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression of trxB, an Spx-dependent gene. Inactivation of ctsR, encoding the regulator of the Clp proteases, reduced trxB expression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted the ctsR phenotype and increased trxB expression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reduced trxB expression. Finally, we show that Spx is itself a regulator of the ctsR operon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity in B. subtilis IMPORTANCE In Bacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of the ctsR operon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.
Project description:Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.
Project description:McsA is a key modulator of stress response in Staphylococcus aureus that contains four CXXC potential metal-binding motifs at the N-terminal. Staphylococcus aureus ctsR operon encodes ctsR, clpC, and putative mcsA and mcsB genes. The expression of the ctsR operon in S. aureus was shown to be induced in response to various types of heavy metals such as copper and cadmium. McsA was cloned and overexpressed, and purified product was tested for metal-binding activity. The protein bound to Cu(II), Zn(II), Co(II), and Cd(II). No binding with any heavy metal except copper was found when we performed site-directed mutagenesis of Cys residues of three CXXC motifs of McsA. These data suggest that two conserved cysteine ligands provided by one CXXC motif are required to bind copper ions. In addition, using a bacterial two-hybrid system, McsA was found to be able to bind to McsB and CtsR of S. aureus and the CXXC motif was needed for the binding. This indicates that the Cys residues in the CXXC motif are involved in metal binding and protein interaction.
Project description:During the development of transformability (competence), Bacillus subtilis synthesizes a set of proteins that mediate both the uptake of DNA at the cell poles and the recombination of this DNA with the resident chromosome. Most, if not all, of these Com proteins localize to the poles of the cell, where they associate with one another, and are then seen to delocalize as transformability declines. In this study, we use fluorescence microscopy to analyse the localization and delocalization processes. We show that localization most likely occurs by a diffusion-capture mechanism, not requiring metabolic energy, whereas delocalization is prevented in the presence of sodium azide. The kinetics of localization suggest that this process requires the synthesis of a critical protein or set of proteins, which are needed to anchor the Com protein complex to the poles. We further show that the protein kinase proteins McsA and McsB are needed for delocalization, as are ClpP and either of the AAA(+) (ATPases associated with a variety of cellular activities) proteins ClpC or ClpE. Of these proteins, at least McsB, ClpC and ClpP localize to the cell poles of competent cells. Our evidence strongly suggests that delocalization depends on the degradation of the postulated anchor protein(s) by the McsA-McsB-(ClpC or ClpE)-ClpP protease in an ATP-dependent process that involves the autophosphorylation of McsB. The extent of cell-pole association at any given time reflects the relative rates of localization and delocalization. The kinetics of this dynamic process differs for individual Com proteins, with the DNA-binding proteins SsbB and DprA exhibiting less net localization.
Project description:The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B.?anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B.?anthracis and open up further interest on this operon, which might be of importance to success of B.?anthracis as pathogen.
Project description:Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.
Project description:Protein quality networks are required for the maintenance of proper protein homeostasis and essential for viability and growth of all living organisms. Hence, regulation and coordination of these networks are critical for survival during stress as well as for virulence of pathogenic species. In low GC, Gram-positive bacteria central protein quality networks are under the control of the global repressor CtsR. Here, we provide evidence that CtsR activity during heat stress is mediated by intrinsic heat sensing through a glycine-rich loop, probably in all Gram-positive species. Moreover, a function for the recently identified arginine kinase McsB is confirmed, however, not for initial inactivation and dissociation of CtsR from the DNA, but for heat-dependent auto-activation of McsB as an adaptor for ClpCP-mediated degradation of CtsR.
Project description:Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.