ctsR of Lactococcus lactis encodes a negative regulator of clp gene expression

Pekka Varmanen1, Hanne Ingmer2 and Finn K. Vogensen1

The Royal Veterinary and Agricultural University, Department of Dairy and Food Science, DK-1958 Frederiksberg C, Denmark1
The Royal Veterinary and Agricultural University, Department of Veterinary Microbiology, DK-1870 Frederiksberg C, Denmark2

Author for correspondence: Finn K. Vogensen. Tel: +45 35 28 32 11. Fax: +45 35 28 32 31. e-mail: fkv{at}biobase.dk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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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 Gram-positive bacteria reflects functional heat-shock regulatory systems.

Keywords: lactic acid bacteria, stress response, heat shock, clp, transcriptional regulation

Abbreviations: 2-D, two-dimensional

The GenBank accession numbers for the nucleotide sequences of ctsR and ORF555 and their flanking regions are AJ249133 and AJ249134, respectively.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Rapid changes in gene expression in response to changes in the environment enable cells to survive stress conditions. In bacteria, transcriptional regulators and alternative {sigma} factors play a crucial role in the process whereby external signals promote dramatic alterations in gene expression.

In Escherichia coli, the alternative sigma factors {sigma}32, {sigma}E and {sigma}54 positively regulate heat-shock-induced gene expression (Bukau, 1993 ; Narberhouse, 1999 ). Among the Gram-positive bacteria, the regulation of gene expression by stress has been well-characterized in Bacillus subtilis. In this soil bacterium, four classes of heat-shock-regulated genes have been identified (Derré et al., 1999a ; Hecker et al., 1996 ). Class I genes are controlled by the HrcA repressor, which binds to the CIRCE operator sequence found in front of genes encoding classical chaperones such as DnaK, GroES and GroEL (Hecker et al., 1996 ). The expression of Class II genes is dependent on the {sigma}B sigma factor whose synthesis and activity are increased under various stress conditions, including heat shock (Hecker et al., 1996 ). Class III genes were recently shown to be negatively regulated by CtsR, which recognizes a directly repeated heptanucleotide operator sequence located in the promoter region of target genes (Derré et al., 1999a ; Krüger & Hecker, 1998 ). Class IV heat-shock genes in B. subtilis are defined as those that are expressed independently of {sigma}B and are devoid of CIRCE or CtsR operator sequences (Derré et al., 1999a , b ). These classes appear to be at least in part conserved in other Gram-positive bacteria since HrcA homologous proteins have been demonstrated in Streptococcus mutans (Jayaraman et al., 1997 ), whilst in Listeria monocytogenes a general stress sigma factor B responds to acid and osmotic stress (Becker et al., 1998 ; Wiedmann et al., 1998 ). Thorough comparison with sequence databases, including unfinished bacterial genome sequences and the unpublished genome sequence of Lactococcus lactis IL1403 revealed that, in addition to B. subtilis, CtsR homologues are present in many Gram-positive bacteria, including Lactococcus (Derré et al., 1999a ). Additionally, a database search for CtsR DNA-binding sites revealed that they are found highly conserved in a wide range of other Gram-positive bacteria, suggesting that CtsR homologous proteins control gene expression in these organisms (Derré et al., 1999a ).

In B. subtilis, the CtsR regulon includes genes encoding products with homology to the highly conserved family of Clp ATPases (ClpC, ClpE) in addition to clpP encoding the ClpP protease (Derré et al., 1999a , b ). Several Clp proteins are chaperones that can also act as ATPase subunits of the ATP-dependent Clp protease complex by associating with the proteolytic component, ClpP. By itself, ClpP, the catalytic component of Clp protease, is able to degrade short peptides; however, to degrade larger substrates, it must associate with one of the Clp ATPases (Maurizi et al., 1990 ).

L. lactis is a Gram-positive bacterium that is widely used in mesophilic starter cultures in the production of a variety of dairy products. In this organism we have recently identified and characterized the clpB, clpC, clpE and clpP genes (Frees & Ingmer, 1999 ; Ingmer et al., 1999 ). Northern blot analysis showed that expression of all of these genes is induced by heat stress, and additionally we found that ClpP is required for survival at high temperature (Frees & Ingmer, 1999 ). Here, we report identification of a gene located just upstream of clpC which encodes a L. lactis homologue of CtsR. By constructing and analysing a ctsR deletion mutant in L. lactis strain MG1363 we furthermore show that CtsR controls the expression of clpP, clpC, clpE and clpB. This is the first report describing the function of a CtsR homologue as a negative regulator of heat-shock-induced gene expression in a bacterium other than B. subtilis.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains and growth media.
L. lactis strains were grown in M17 (Terzaghi & Sandine, 1975 ) supplemented with 0·5% glucose (GM17) or in minimal MOPS-based SA medium (Jensen & Hammer, 1993 ). E. coli XL-1 Blue (Stratagene) and JM-105 (Amersham Pharmacia Biotech) were grown in LB medium. When needed, ampicillin (50 µg ml-1) or tetracycline (8 µg ml-1 for E. coli and 2 µg ml-1 for L. lactis) were added.

General DNA techniques, transformation and DNA amplification.
Molecular cloning techniques were performed essentially as described by Sambrook et al. (1989) . Restriction enzymes, T4 DNA ligase and deoxyribonucleotides were obtained from Roche or New England Biolabs and were used according to the instructions of the suppliers. Chromosomal DNA isolation from and transformation of L. lactis were performed essentially as described previously (Arnau et al., 1996 ). PCR amplification was performed using the DynaZyme DNA polymerase as described by the manufacturer (Finnzymes). PCR products were purified with the Qiagen PCR purification kit.

Sequencing and sequence analysis of the upstream and downstream regions of clpC.
An inverse PCR strategy was utilized to obtain DNA sequence of the upstream region of clpC. Briefly, 1 µg chromosomal DNA from L. lactis MG1363 was digested with DraI. One-tenth of the digested DNA was ligated in a volume of 100 µl and used as template in PCR with primers p1 and p2 (Table 1; Fig. 1). The resulting 1·4 kb PCR product was partially sequenced using primers p3, p4 and p5 (Table 1).


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Table 1. Oligonucleotide primers used in PCR amplification, sequencing and primer extension

 


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Fig. 1. Partial restriction map of the L. lactis MG1363 ctsR–clpC region. The positions and orientations of ctsR (453 bp), clpC (2445 bp) and ORF555 (555 bp) are shown. The binding sites of primer pairs p1/p2 and p6/p7, used in inverse PCR, are indicated. The location of the inverted repeat structure representing a putative transcription terminator is marked with a hairpin.

 
The DNA sequence downstream of clpC was also obtained by inverse PCR. A single 2 kb product resulted from PCR of EcoRI-digested chromosomal DNA using p6 and p7 (Table 1) as primers. Sequence analysis of this fragment was performed using primers p8 and p9 (Table 1). The sequencing reactions were carried out using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit from Amersham Pharmacia Biotech and analysed using an ALFexpress DNA sequencer (Amersham Pharmacia Biotech). Both DNA strands were sequenced using sequence-specific oligonucleotides. The GCG (version 9.1; University of Wisconsin Genetics Computer Group) and the DNASIS (Hitachi Software) software packages were used for assembling and analyses of DNA sequences. Sequence comparisons with the GenBank database were accomplished using the National Center for Biotechnology Information (NCBI) BLAST2 network server at http://www.ncbi.nlm.nih.gov/blast. Comparison with the Unfinished Microbial Genomes database was perfomed using the NCBI BLAST server at http://www.ncbi.nlm.nih.gov/blast/unfinishedgenome.html.

Construction of chromosomal deletion mutants of ctsR.
A replacement recombination technique was used to construct two recombinant strains of L. lactis MG1363 carrying different sized in-frame deletions of ctsR. Gene replacement vectors were constructed using the plasmid pG+host8 with a thermosensitive origin of replication (Biswas et al., 1993 ). For construction of a strain with a 291 bp deletion of ctsR, a 800 bp DNA fragment carrying the upstream region and first 70 nucleotides of the ctsR gene was generated by PCR using primers p10 and p11 (Table 1). A 1000 bp fragment carrying the downstream region and the last 87 nucleotides of the ctsR gene was amplified using the primers p12 and p13 (Table 1). The 800 bp and 1000 bp PCR-generated products were digested with XbaI/PstI and PstI/SalI, respectively, and subsequently ligated with pG+host8 digested with XbaI/SalI. The resulting plasmid carrying a 1·8 kb insert was named pPV10. To obtain another vector that could be used to make a strain with a 168 bp deletion in ctsR the XbaI–PstI fragment of pPV10 was replaced with a XbaI/PstI-digested 600 bp PCR fragment generated by PCR using primers p14 and p15 (Table 1). The resulting integration vector carrying a 1·6 kb insert was named pPV11.

For integration of replacement vectors, transformed, tetracycline-resistant L. lactis colonies were grown overnight at 37 °C in GM17 broth (containing 2 µg tetracycline ml-1) and plated on GM17 agar (containing 2 µg tetracycline ml-1), followed by incubation overnight at 37 °C. To allow excision of the integrated vectors from the chromosome, the integrants were grown overnight at 28 °C and plated on GM17 agar including tetracycline (2 µg ml-1) followed by incubation at 28 °C. The excised plasmid was cured by incubating the strains at 37 °C in GM17 without antibiotic. Tetracycline-sensitive colonies were tested by PCR for the presence of the wild-type gene or a gene carrying an internal deletion.

For gene replacement with pPV10, 19 colonies were tested and 2 of them gave a single 1·5 kb PCR product corresponding to the ctsR deletion, whereas a 1·8 kb product was amplified from the control MG1363 strain (data not shown). One of the two mutant strains was named PV1 and was chosen for further characterization. One of the ten colonies tested after replacement recombination with pPV11 gave a single 1·6 kb PCR product. The mutant strain was named PV2. The PV1 and PV2 strains carry a 291 bp and a 168 bp in-frame deletion in the ctsR gene and are capable of encoding a 54 amino acid and a 95 amino acid recombinant CtsR protein, respectively. The CtsR encoded by PV1 lacks amino acid residues 24–122 including the probable helix–turn–helix motif (amino acids 29–48) in the wild-type CtsR. The amino acids in positions 65–122 in wild-type CtsR are not present in CtsR of PV2.

RNA methods.
For preparation of total RNA from L. lactis MG1363, PV1 or PV2, cells were grown at 30 °C in defined GSA medium to an OD450 of 0·4–0·5 after which heat stress was applied by transferring the tubes to 43 °C. Cell samples were taken 0 and 10 min after the heat shock. Harvested cells were disrupted with glass beads (106 µm and less in diameter; Sigma) in a homogenizer (Fastprep FB 120; Savant) for 45 s. Total RNA was subsequently purified with an RNeasy mini kit (Qiagen) according to the instructions of the supplier. RNA gel electrophoresis was according to Pelle & Murphy (1993) . Samples were transferred by vacuum blotting onto positively charged nylon membrane and hybridized at 65 °C using a buffer containing 4xSSC, 50 mM sodium phosphate pH 6·8, 5xDenhardt solution, 0·2% SDS, 200 µg salmon sperm DNA ml-1 and the appropriate probe DNA. The clpC-, clpP-, clpE- and clpB-specific probes were obtained by PCR using primer pairs p16/p17, p18/p19, p20/p21 and p22/p23 (Table 1), respectively. The probe specific for the clpC-downstream region was obtained with primers p24 and p25 (Table 1). The hybridization probes were labelled with [{alpha}-32P]dATP [>3000 Ci mmol-1 (>111 TBq mmol-1); Amersham]. Following hybridization, the membranes were scanned and the signal quantified using a PhosphorImager (Storm system; Molecular Dynamics) and ImageQuaNT (version 4.2; Molecular Dynamics). The total amount of RNA present on the membrane was evaluated by hybridizing with a probe specific for L. lactis 16S rRNA obtained by PCR with primers p26 and p27 (Table 1).

Primer extension analysis was performed using 1·5–10 µg total RNA and an ALF DNA sequencer (Amersham Pharmacia Biotech) essentially as described by Myöhänen & Wahlfors (1993) . The anti-sense fluorescein-labelled oligonucleotide used in primer extension was p28 (Table 1).

Two-dimensional (2-D) PAGE.
L. lactis MG1363 (wild-type) and PV1 ({Delta}ctsR) were grown in defined GSA media with a low methionine concentration (5 µg ml-1). Labelling of proteins with [35S]methionine and 2-D protein gel electrophoresis were according to Kilstrup et al. (1997) .

Physiological characterization of L. lactis strains.
The growth rates of L. lactis MG1363, PV1 and PV2 at 30 °C were determined by diluting overnight cultures 1:1000 in prewarmed GM17 and following growth by measuring OD600. The ability of the strains to form colonies at 37 °C was tested by diluting overnight cultures 1000-fold in GM17 and growing the cells at 30 °C to an OD600 of 0·4. Appropriate dilutions were spread on prewarmed GM17 plates and incubated at 37 °C. Analysis of thermotolerance was essentially as described previously (Koch et al., 1998 ). Briefly, overnight cultures were diluted 1000-fold in GM17 and grown at 30 °C to an OD600 of 0·4, at which point 3 ml samples were harvested and resuspended in 3 ml GM17 in glass tubes with a diameter of 1 cm. After 30 min incubation in a 30 or 40 °C water bath, the tubes were placed in a 53 °C water bath and c.f.u. were determined after 0, 15, 30, 45 and 60 min. The ability of the strains to grow at 10 °C was tested by plating samples from cultures growing exponentially in GM17 at 30 °C and incubating the plates at 10 °C. The appearance of colonies was followed after 1, 2, 3, 4 and 5 d incubation. Salt sensitivity of MG1363 and PV1 was tested by plating samples from cultures growing exponentially in GM17 at 30 °C onto GM17 agar containing 4·0 or 5·0% NaCl. Plates were incubated for 3 d at 30 °C and monitored for growth.


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INTRODUCTION
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DISCUSSION
REFERENCES
 
Sequence analysis of the upstream and downstream regions of clpC
In a previous study we identified genes encoding ClpB-, ClpC-, ClpE- and ClpP-homologous proteins (Frees & Ingmer, 1999 ; Ingmer et al., 1999 ). Whilst analysing the DNA region upstream of clpC we obtained a 1·4 kb fragment by inverse PCR. Sequence analysis of this product showed that a 453 bp ORF was present capable of encoding a 17 kDa protein product (see Fig. 1). The amino acid sequence of the 17 kDa product showed 38% identity with the recently characterized CtsR from B. subtilis that negatively regulates the transcription of several stress-regulated genes (Derré et al., 1999a ; Hecker et al., 1996 ). Thus the 453 bp L. lactis ORF was named ctsR. Comparison with the Unfinished Microbial Genomes database revealed that the potential CtsR homologues in Streptococcus mutans, Streptococcus pyogenes, Enterococcus faecalis and Streptococcus pneumoniae share 51, 49, 48 and 47% identical amino acid residues with the L. lactis CtsR, respectively. The L. lactis CtsR protein contains a predicted helix–turn–helix DNA-binding motif also present in other CtsR homologues (Derré et al., 1999a ). The stop codon (TGA) of the L. lactis ctsR overlaps the probable start site of clpC (Ingmer et al., 1999 ) by 8 nucleotides. A typical prokaryotic RBS (AAGGA) is present in the correct position and a putative promoter region (TTGGTA-17 nt-TATAAT) is located 54 nucleotides upstream of the ctsR start codon. Immediately downstream of the putative promoter region we found a 17 bp sequence (AGTCAGTAATAGTCAAA) closely resembling the suggested consensus sequence, A/GGTCAAANANA/GGTCAAA, of the CtsR-binding site in Gram-positive bacteria (Derré et al., 1999a ). Upstream of ctsR we found a DNA region with 99% identity to the 3' ends of L. lactis insertion elements (GenBank accession numbers X78469, X52273, M27276 and X53013). Downstream of ctsR we did not identify an inverted repeat structure typical of Rho-independent transcriptional terminators, suggesting that ctsR and clpC are part of the same operon. Sequence analysis of the DNA region downstream of clpC revealed a start codon of a 555 bp ORF (ORF555) located 139 bp downstream of clpC. ORF555 is preceded by a putative RBS and could encode a 21 kDa protein with 45% identity to the gene product of B. subtilis yvyD (Drzewiecki et al., 1998 ) and 30–37% identity to {sigma}54 modulating factors of various Gram-negative bacteria (Merrick & Coppard, 1989 ). A putative Rho-independent transcription terminator is located 5 nucleotides downstream of the ORF555 stop codon.

ctsR and clpC are part of a tricistronic operon in L. lactis
Expression of the L. lactis ctsR was analysed by Northern blotting using RNA isolated from L. lactis MG1363 (wild-type) cells and a 0·8 kb clpC-specific PCR fragment as a probe. The 3·4 kb transcript detected in MG1363 (Fig. 2a, lanes 1–2) suggests that ctsR and clpC are co-transcribed. Additionally, Northern blot analysis of a mutant carrying a 291 bp in-frame deletion within ctsR (PV1, see Methods) showed that the transcript size was reduced to 3·1 kb (Fig. 2a, lanes 3–4), thus confirming that ctsR and clpC are part of the same operon. Since the size of the transcript detected with the clpC-specific probe is slightly longer than the size expected of a transcript covering only ctsR and clpC, we additionally hybridized with a probe located downstream of clpC covering ORF555. This probe also reacted with a 3·4 kb and 3·1 kb transcript in RNA isolated from MG1363 and PV1 cells, respectively (data not shown), demonstrating that ORF555 is part of the ctsR operon. Primer extension mapping of the 5' end of the transcript indicated that the transcription initiation site is located 50 nucleotides upstream of the ctsR start codon (data not shown).



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Fig. 2. Heat-shock-induced expression of clpC (a), clpP (b), clpE (c) and clpB (d) in L. lactis MG1363, PV1 and PV2. Total RNA was isolated from MG1363 (lanes 1 and 2), PV1 (lanes 3 and 4) and PV2 (lanes 5 and 6) cells growing exponentially at 30 °C (lanes 1, 3 and 5) or 10 min after transfer to 43 °C (lanes 2, 4 and 6). Northern blot analyses were performed with clpC-, clpP-, clpE- and clpB-specific DNA probes as described in Methods. The numbers on the left indicate the sizes of RNA molecular mass markers (Gibco-BRL). The bar diagrams show the relative mRNA induction ratios calculated by dividing the signal from RNA of the mutants by the signal from RNA of wild-type MG1363 at 30 °C.

 
Expression of clpC, clpB, clpE and clpP is negatively regulated by ctsR
The L. lactis PV1 strain encodes a CtsR protein in which the central 99 amino acids are deleted, including the predicted helix–turn–helix motif of the wild-type. Since the CtsR regulon in B. subtilis comprises several clp genes, we investigated the expression of the L. lactis clpB, clpC, clpE and clpP in the wild-type (MG1363) as well as in the ctsR-deleted strain (PV1) by Northern blot analysis. Fig. 2(a–d, lane 3) shows that the amounts of clpC, clpP, clpE and clpB transcripts are elevated in the PV1 strain (lane 3) at 30 °C compared to those present in the wild-type strain (lane 1). When the PV1 strain was shifted to 43 °C for 10 min there was no further increase in the amount of clpP-specific transcript (Fig. 2b, lane 4) whilst the amounts of clpC, clpE and clpB mRNA were somewhat increased when compared to the amounts present at 30 °C (Fig. 2a, c, d, lane 4).

In a study of CtsR from B. subtilis it has been suggested that the helix–turn–helix domain located in the N-terminal part of the protein is important in transcriptional repression (Derré et al., 1999a ). To address the role of the C-terminal part of the L. lactis CtsR protein, we constructed a mutant (PV2) producing a CtsR that lacks amino acids 65–122. In this strain CtsR-mediated repression of clp gene expression was only partially relieved and, furthermore, the transcription of all the genes studied was heat-inducible (Fig. 2a–d, lanes 5–6).

At the protein level, we investigated the expression of the clp-encoded products in PV1 and MG1363 at 30 °C by separating pulse-labelled [35S]methionine proteins using 2-D protein gel electrophoresis. We found that two proteins, ClpP (Hsp-23) and ClpE (Hsp-85), previously identified and located on 2-D protein gels (Kilstrup et al., 1997 ; Frees & Ingmer, 1999 ; Ingmer et al., 1999 ) were synthesized at a clearly elevated rate in PV1 compared to MG1363 (Fig. 3) whilst we were unable to detect the two clpB-encoded products (Ingmer et al., 1999 ). Additionally, three unidentified protein spots (arrows in Fig. 3b) were also synthesized at an increased rate in the ctsR-deleted PV1 strain compared to the wild-type strain.



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Fig. 3. Protein expression in L. lactis MG1363 (wild-type) (a) and PV1 ({Delta}ctsR) (b). Cells were grown exponentially in GSA medium and pulse-labelled with [35S]methionine for 10 min at 30 °C. Equal amounts of protein were analysed by 2-D PAGE. Positions of ClpE and ClpP are indicated. Three additional, unknown proteins induced in PV1 are indicated by arrows.

 
Physiological characterization of ctsR deletion strains
The mean growth rates of wild-type L. lactis (MG1363) cells and of the ctsR deletion strains (PV1 and PV2) were determined in complex medium (GM17) at 30 °C. The results of three independent experiments showed that the mean growth rates of MG1363, PV1 and PV2 were 28·5±1·0, 31·9±0·5 and 31·0±0·8 min, respectively. Since the PV1 and PV2 strains showed increased expression of several of the heat-inducible clp genes, we tested whether this provided the strains with increased tolerance of exposure to lethal temperatures when compared to MG1363. However, when cells that were growing exponentially at 30 °C were challenged for 15 min at 53 °C the number of surviving cells was not significantly greater for PV1 or PV2 than MG1363 (data not shown). Additionally, thermotolerance was induced in all strains when the cells were preheated for 30 min at 40 °C before the incubation at 53 °C when the wild-type strain gave a slightly greater number of c.f.u. compared to the mutant strains (data not shown). The ability to grow at 37 °C was tested by plating exponentially growing cells on GM17 plates as described in Methods. Both wild-type and mutant strains were able to form colonies at 37 °C. However, 2 d incubation was required for MG1363 strain to form colonies with a mean size of 1 mm whereas 3 d incubation was needed for both PV1 and PV2 strains. Furthermore, we tested whether the larger deletion of ctsR resulted in an altered resistance to either cold (10 °C), salt (4 or 5% NaCl) or puromycin (20 µg ml-1) treatments but did not find an effect of the deletion when compared to the wild-type strain (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Recently, several clp genes have been characterized in L. lactis (Frees & Ingmer, 1999 ; Ingmer et al., 1999 ). In L. lactis, ClpP is required for survival at high temperature and for growth in the presence of puromycin (Frees & Ingmer, 1999 ). Additionally, three genes have been identified, named clpB, clpC and clpE, which encode products with homology to the Clp family of ATPases. In contrast to E. coli, in which the regulation of clpP and clp ATPase genes have been studied extensively (Bukau, 1993,bibr id=4>), very little was known about the regulation of Clp expression in Gram-positive bacteria. However, recently, two studies of B. subtilis showed that the first gene of the clpC operon, designated ctsR (class three stress gene regulator), encodes a negative regulator of class III heat-shock genes (Derré et al., 1999a ; Hecker et al., 1996 ). In the present work, we have sequenced and characterized the gene encoding the CtsR homologue of L. lactis MG1363. As in B. subtilis, ctsR of L. lactis is the first gene of the clpC operon. In B. subtilis the clpC operon consists of six genes (Krüger & Hecker, 1998 ), whereas in L. lactis MG1363 there are only three genes, ctsR, clpC and ORF555, expressed via the same mRNA driven from the promoter upstream of ctsR. ORF555 encodes a protein with 45% identity to the yvyD gene product from B. subtilis, which recently was shown to be induced by several stress conditions, including amino acid depletion (Drzewiecki et al., 1998 ). Interestingly, yvyD is a member of both {sigma}B and {sigma}H regulons whilst its gene product negatively regulates the activity of the {sigma}L regulon. However, the exact function of the protein is still unknown (Drzewiecki et al., 1998 ). It remains to be elucidated whether the YvyD homologue has any regulatory role in L. lactis.

When we constructed an in-frame deletion of ctsR covering the region encoding the putative DNA-binding helix–turn–helix motif we found that in the absence of stress the amounts of clpC, clpP, clpE and clpB transcripts were elevated compared to the amounts present in wild-type cells. At the protein level, we additionally found that ClpP and ClpE were synthesized at an increased rate in the ctsR-deleted strain compared to the wild-type strain. The ClpC protein remained unidentified on 2-D PAGE gels (Kilstrup et al., 1997 ; Ingmer et al., 1999 ) and surprisingly, we were unable to detect the two clpB-encoded protein products in PV1. The reason for this remains unknown. However, although the clpB-specific hybridization signal obtained from RNA isolated from non-stressed cells of PV1 clearly was greater than that obtained from non-stressed wild-type cells, the signal appeared smeary compared to that obtained with the other clp-specific probes using the same RNA. The smeary signal could be a result of rapid degradation of clpB transcript under non-stressed conditions.

When we compared the expression of the different clp genes in the ctsR mutant strain PV1 grown at 30 °C, we found that the amount of clpP-specific transcripts had not increased after 10 min (this study) or 15 min (unpublished data) exposure to 43 °C, suggesting that the internally deleted CtsR protein in this strain is inactive. Furthermore, this result suggests that, in contrast to B. subtilis (Derré et al., 1999a ), in L. lactis there is no additional regulation of clpP expression under heat shock. Interestingly, in the PV2 strain, in which only amino acids 65–122 are deleted from CtsR, clpP expression was still partially repressed at 30 °C and could be induced at 43 °C. This suggests that the truncated CtsR protein synthesized in PV2 retains part of its ability to bind DNA and furthermore is able to sense changes in temperature. It has been suggested that the B. subtilis CtsR protein acts as a temperature-sensing protein (Derré et al., 1999a ). If the L. lactis CtsR has the same activity, our results suggest that this function is located either within the first 65 or the last 29 amino acids of the protein. It should be noted that the truncated CtsR encoded by PV2 contains two additional alanine residues at the site of internal deletion, which may also contribute to the partial loss of activity. However, this does not change the hypothesis for the location of the temperature-sensing function in CtsR of L. lactis.

In contrast to the clpP transcript, the amounts of clpB and clpE transcripts were increased in repeated experiments in which PV1 was exposed to 43 °C. Thus, our data indicate that heat-shock-induced expression of clpB and clpE is only in part regulated by CtsR in lactococci. In B. subtilis, the expression of the CtsR regulon (clpP, clpC and clpE) also appears to be controlled by an additional regulator, but in both B. subtilis and L. lactis this regulator remains unidentified. The low expression level of clpC compared to other clp genes in L. lactis hampers the assessment of the presence of possible additional regulation under heat shock.

It has previously been shown that the dnaK mutant of L. lactis is more thermosensitive than the wild-type and, furthermore, that dnaK is important in developing thermotolerance when cells are pre-treated at 40 °C before lethal exposure to 53 °C (Koch et al., 1998 ). Also, the clpP mutation led to increased sensitivity to elevated temperatures (Frees & Ingmer, 1999 ). In this paper, we have shown that in the PV1 and PV2 strains, the expression of clpP and the other clp genes are elevated in cells growing in GSA (this study) and in GM17 (unpublished). However, PV1 and PV2 do not show greater thermotolerance than the wild-type, indicating that clpP and the other genes regulated by CtsR may not be as important as dnaK in developing heat-induced thermotolerance in L. lactis.

In a study of B. subtilis CtsR DNA-binding activity, Derré et al. (1999a) demonstrated that purified CtsR recognizes and binds to a directly repeated heptanucleotide sequence, A/GGTCAAA/T, separated by three nucleotides. Based on a compilation of 32 putative CtsR-binding sites, the following consensus sequence was proposed: A/GGTCAAANANA/GGTCAAA (Derré et al., 1999a ). In L. lactis, the putative operator sites of CtsR are located immediately downstream of the promoter for the ctsR–clpC–ORF555 operon, overlapping the -35 region of the clpP promoter and that of the putative clpB promoter and 10 nucleotides downstream of the putative clpE promoter (Fig. 4). The CtsR boxes in front of clpP and clpB have an orientation inverse to that of the consensus sequence. The probable CtsR-binding site in the upstream region of clpE (Fig. 4) is separated by 490 nucleotides from the start codon of clpE. An uncharacterized ORF of 315 bp is located between the CtsR-binding site and clpE. Our results presented here (Fig. 2) suggest that the size of the clpE transcript is 2·9 kb, which is in accordance with the predicted size of a bicistronic operon consisting of a 315 bp ORF and clpE. The sequences of the probable CtsR-binding sites in front of clpE and clpP match exactly the suggested consensus, whereas the nearly identical sites in front of clpB and clpC (Fig. 4) (G/AGTCAgtAATAGTCAAA) have a 2 bp mismatch compared to the consensus. However, it remains to be elucidated whether these differences in the binding regions have any consequences in the regulation mediated by CtsR.



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Fig. 4. Promoter regions of ctsR–clpC and clpP, and putative promoter regions of clpE and clpB. The predicted -35 and -10 hexanucleotides are in bold. The 5' ends of ctsR–clpC- and clpP-specific mRNAs found by primer extension are shown by vertical arrows. The probable operator sites for CtsR are boxed.

 

   ACKNOWLEDGEMENTS
 
We are grateful to our project partners, K. Hammer, M. Kilstrup and A. K. Nielsen, for helpful discussions throughout the work. D. Frees is acknowledged for fruitful discussions and help with the 2-D PAGE gels. We are grateful to D. O. Jensen and C. Rasmussen for excellent technical assistance. This work was financed by FØTEK and MFF (Danish Dairy Research Board).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 12 November 1999; revised 24 February 2000; accepted 13 March 2000.