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
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ABSTRACT |
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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.
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INTRODUCTION |
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In Escherichia coli, the alternative sigma factors 32,
E and
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
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
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.
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METHODS |
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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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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 XbaIPstI 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 24122 including the probable helixturnhelix motif (amino acids 2948) in the wild-type CtsR. The amino acids in positions 65122 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 0·40·5 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 [
-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 1·510 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 (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)
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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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RESULTS |
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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 12) 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 34), 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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In a study of CtsR from B. subtilis it has been suggested that the helixturnhelix 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 65122. 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 56).
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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DISCUSSION |
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When we constructed an in-frame deletion of ctsR covering the region encoding the putative DNA-binding helixturnhelix 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 65122 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 ctsRclpCORF555 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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ACKNOWLEDGEMENTS |
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Received 12 November 1999;
revised 24 February 2000;
accepted 13 March 2000.