The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance,stress resistance,motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.     

Escherichia coli YaeJ protein mediates a novel ribosome-rescue pathway distinct from SsrA- and ArfA-mediated pathways

CtsR, the global heat shock repressor in low GC, Gram+ bacteria, regulates a crucial subset of genes involved in protein quality control. CtsR de-repression occurs not only during heat stress but also during a variety of other environmental stresses, most notably thiol-specific oxidative stress. Here we report that McsA acts as a molecular redox switch that regulates CtsR de-repression via the activation of McsB. Once critical thiols of McsA become oxidized, the strong interaction between McsA and McsB is interrupted and free McsB is no longer inhibited by McsA, resulting in the inactivation of CtsR. This mechanism differs significantly from inactivation of CtsR during heat stress demonstrating a dual activity control of CtsR. Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation.     

CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram-positive bacteria

clpP and clpC of Bacillus subtilis encode subunits of the Clp ATP-dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR , the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC , as well as clpE , which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNaseI footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram-positive bacteria, including Listeria monocytogenes , Streptococcus salivarius , S. pneumoniae , S. pyogenes , S. thermophilus , Enterococcus faecalis , Staphylococcus aureus , Leuconostoc oenos , Lactobacillus sake , Lactococcus lactis and Clostridium acetobutylicum . CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram-positive bacteria.     

A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis

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.     

CtsR, the Gram-positive master regulator of protein quality control, feels the heat

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.     

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.