ctsR of Lactococcus lactis encodes a negative regulator of clp gene expression (original) (raw)
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Microbiology, 2000
Bacteria undergo a complex programme of differential gene expression in response to stress. In Bacillus subtilis, it was recently shown that CtsR, a negative transcriptional regulator, mediates stress-induced expression of components of the Clp protease complex. In this study, a gene was identified in the Gram-positive bacterium Lactococcus lactis that encodes a 17 kDa product with 38 % identity to the CtsR protein of B. subtilis. By Northern analyses it was found that in a L. lactis strain carrying a large internal deletion of ctsR, including the region encoding a putative helix-turn-helix motif, the amounts of clpC, clpP, clpB and clpE mRNAs were increased 3-8-fold compared to those present in wild-type L. lactis MG1363. In another ctsR mutant strain in which only one-third of CtsR was deleted, leaving the putative DNA-binding domain and the C-terminal 29 amino acids intact, only minor derepression of clp gene expression was observed and, furthermore, all the clp genes were still induced by heat. These results indicate that the amino acids of CtsR involved in temperature sensing are located either close to the DNA-binding domain or in the C-terminal part of the protein. Thus, in L. lactis in addition to B. subtilis, CtsR is a key regulator of heat-shock-induced gene expression, suggesting that the presence of CtsR-homologous DNA-binding sites observed in many Grampositive bacteria reflects functional heat-shock regulatory systems.
ClpE from Lactococcus lactis Promotes Repression of CtsR-Dependent Gene Expression
Journal of Bacteriology, 2003
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 ClpPdependent 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.
Journal of Bacteriology, 1998
The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either ς B or a class III stress induction mechanism acting at a ς A -like promoter. When the clpC operon was placed under the control of the isopropyl-β- d -thiogalactopyranoside (IPTG)-inducible P spac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR , containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of ς B , suggesting a second induction ...
Analysis of heat shock gene expression in Lactococcus lactis MG1363
Microbiology, 1996
The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnal and gm€L homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnal and gm€LS after transfer from 30 "C to 43 "C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, off7 and g @ , resembled the pattern observed for the above genes, although maximum induction was observed earlier for off7 and grlpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for fcsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named odd, was shown not to be regulated by heat shock.
Molecular Microbiology, 2001
Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.
The Lactobacillus plantarum ftsH Gene Is a Novel Member of the CtsR Stress Response Regulon
Journal of Bacteriology, 2009
FtsH proteins have dual chaperone-protease activities and are involved in protein quality control under stress conditions. Although the functional role of FtsH proteins has been clearly established, the regulatory mechanisms controlling ftsH expression in gram-positive bacteria remain largely unknown. Here we show that ftsH of Lactobacillus plantarum WCFS1 is transiently induced at the transcriptional level upon a temperature upshift. In addition, disruption of ftsH negatively affected the growth of L. plantarum at high temperatures. Sequence analysis and mapping of the ftsH transcriptional start site revealed a potential operator sequence for the CtsR repressor, partially overlapping the ؊35 sequence of the ftsH promoter. In order to verify whether CtsR is able to recognize and bind the ftsH promoter, CtsR proteins of Bacillus subtilis and L. plantarum were overproduced, purified, and used in DNA binding assays. CtsR from both species bound specifically to the ftsH promoter, generating a single protein-DNA complex, suggesting that CtsR may control the expression of L. plantarum ftsH. In order to confirm this hypothesis, a ⌬ctsR mutant strain of L. plantarum was generated. Expression of ftsH in the ⌬ctsR mutant strain was strongly upregulated, indicating that ftsH of L. plantarum is negatively controlled by CtsR. This is the first example of an ftsH gene controlled by the CtsR repressor, and the first of the low-G؉C gram-positive bacteria where the regulatory mechanism has been identified.
Applied and Environmental Microbiology, 2000
The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained. Northern blot analysis revealed low abundance of the cspC transcript, indicating that the stability of cspC mRNA is low. The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased. Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of coldinduced proteins of L. lactis, were induced. Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed. Overproduction of CspB and overproduction of CspE result in increased survival when L. lactis is frozen (maximum increases, 10-and 5-fold, respectively, after 4 freeze-thaw cycles). It is concluded that in L. lactis CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both. 3756 on November 3, 2015 by guest http://aem.asm.org/ Downloaded from monitor the physiological and regulatory effects of the CSPs. CspB, CspD, and CspE could be overproduced at high levels, whereas for CspA and CspC only low levels of overproduction were detected, probably due to low protein and mRNA stability at 30°C, respectively. Overproduction of specific CSPs resulted in major induction of other CSPs and CIPs, indicating that these proteins have a regulatory function. L. lactis strains overproducing CspB or CspE did not have a shorter lag time upon cold shock but did show enhanced survival after freezing.
Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance
Journal of Bacteriology, 1994
A member of the clpC subfamily of stress response-related Clp ATPases was cloned from Bacillus subtilis. The B. subtilis clpC gene was induced in response to various stresses, including heat shock. Its product was identified as a general stress protein (Gsp12) described previously. A dramatic increase in the amount of clpC mRNA immediately after exposure to multiple stresses suggested regulation on a transcriptional level. Induction by heat shock was independent of the alternative sigma factor SigB, indicating a new mechanism of heat shock induction in B. subtilis. A clpC insertional mutant had an impaired tolerance for heat shock and salt stress. Furthermore, the mutation triggered the formation of elongated cells, a phenomenon particularly pronounced during stress.
Journal of Bacteriology, 1999
In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes ( clpC , clpE , and clpB ) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX 2 CX 22 CX 2 C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB , clpC , or clpE , we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that resul...
csp-like genes of Lactobacillus delbrueckii ssp. bulgaricus and their response to cold shock
Fems Microbiology Letters - FEMS MICROBIOL LETT, 2003
The two csp-like genes from the lactic acid bacterium Lactobacillus delbrueckii ssp. bulgaricus were characterized and designated cspA and cspB. The gene cspA has been identified using a polymerase chain reaction (PCR)-based approach with degenerated primers and further characterized using an inverse PCR strategy. cspA encodes a protein of 65 amino acid residues which displays between 81 and 77% identity with proteins CspL and CspP of Lactobacillus plantarum. cspB has been identified as a cspA ortholog using the partial sequence of the L. bulgaricus ATCC11842. cspB encodes a protein of 69 amino acids which has 42% identity with CspA. Northern blot analyses showed that cspA is transcribed as a single gene and that its transcription increased after a temperature downshift from 42 to 25°C. In contrast, cspB is part of an operon transcribed at constant level irrespective of the temperature. These results indicate that cspA encodes the only Csp-like protein of L. bulgaricus induced by a ...