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Identification of the DNA-binding protein, HrcA, of Streptococcus thermophilus
FEMS Microbiology Letters 198 (2001) 177^182
www.fems-microbiology.org
Identi¢cation of the DNA-binding protein, HrcA, of Streptococcus thermophilus Luca Martirani a , Ra¡aella Raniello a , Gino Naclerio b , Ezio Ricca a , Maurilio De Felice a; * a
Department of General and Environmental Physiology, Section of Microbiology, University Federico II, via Mezzocannone 16, 80134 Naples, Italy b Faculty of Science, University of Molise, Via Mazzini 8, 86170 Isernia, Italy Received 5 March 2001; received in revised form 15 March 2001; accepted 15 March 2001
Abstract HrcA is a negative transcriptional factor controlling the expression of the stress-specific operons dnaK and groESL in several bacteria. Although the HrcA structural gene has been identified in various organisms, studies at the protein level have been so far limited and mostly restricted to Bacillus subtilis. We have identified the HrcA protein of Streptococcus thermophilus and show here that it is a dimer with a native molecular mass of 74.5 kDa and a sequence-specific DNA-binding activity. Partially denatured and inactive S. thermophilus HrcA recovered its binding activity in the presence of the GroEL chaperone. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : HrcA; DNA-binding protein; Streptococcus thermophilus
1. Introduction In natural environments bacteria often face stressful conditions, like nutrient starvation, temperature-, pH-, oxygen- and osmotic-shocks, that negatively a¡ect cell growth and viability. Bacteria are able to adapt their metabolism to and protect their cellular structures from the stressful conditions with the action of speci¢c stress proteins. Among the stress proteins, particularly important are the products of the dnaK and groESL operons that form the DnaK-DnaJ-GrpE and GroEL-GroES chaperone machines, respectively. These two multiprotein complexes are involved in several cellular processes, like folding of nascent polypeptides, maintaining proteins to be secreted in a secretion-competent form and refolding of partially denatured proteins [1]. In Escherichia coli GroEL also acts as an RNA chaperone which protects mRNA from nucleases [2].
* Corresponding author. Tel. : +39 (81) 2534638; Fax: +39 (81) 5514437; E-mail: [email protected]
The dnaK and groESL operons are normally transcribed at a low basal level and are induced in response to heat shock or to other conditions that increase the cytoplasmic levels of denatured proteins [1,3,4]. Two main transcriptional control mechanisms have been so far described: (i) a positive one, involving the stress-speci¢c transcriptional factor c32 , extensively characterized in E. coli [1], and (ii) a negative one involving the repressor protein HrcA [3]. HrcA is a DNA-binding protein, encoded by hrcA, the promoter proximal gene of the dnaK operon in Bacillus subtilis [5]. The HrcA-binding site is the CIRCE (controlling inverted repeat of chaperone expression) element [6], a palindromic sequence present in the promoter region of the dnaK and groESL operons of several bacteria [3]. In addition, the CIRCE element has been found in the promoter region of the dnaJ gene of Lactococcus lactis [7] and the groESL operon of Streptomyces coelicolor, Streptomyces albus and Bradyrhizobium japonicum [8,9]. Mogk et al. [10] have proposed that in B. subtilis HrcA is kept in an active form by interaction with GroEL. During normal growth, GroEL would stabilize HrcA which, in turn, binds to CIRCE, thus blocking groESL and dnaK transcription. Upon stress induction, accumulation of unfolded proteins within the cell would sequester GroEL, which causes HrcA inactivation and, consequently, active transcription of groESL and dnaK.
0378-1097 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 4 2 - 2
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Here we report the identi¢cation and characterization of HrcA from Streptococcus thermophilus. This organism is a moderately thermophilic lactic acid bacterium (LAB) that, in spite of its extensive biotechnological utilization, has so far been the subject of only limited studies. The understanding of stress response mechanisms in S. thermophilus is of particular interest, since during industrial fermentations this organism is exposed to stress conditions such as media acidi¢cation, temperature changes, high osmotic pressure and changes in oxygen concentration. The presence of HrcA has previously been suggested for other LAB, like L. lactis [7,11], Lactobacillus helveticus [12] and Lactobacillus johnsonii [13], based only on nucleotide sequence homologies and this is to our knowledge the ¢rst report of a direct characterization of HrcA from a LAB. 2. Materials and methods 2.1. Bacterial strains and media S. thermophilus and B. subtilis strains were ST11 [14] and PY79 [15], respectively. S. thermophilus was grown anaerobically at 42³C in M17 medium [16] supplemented with 1% (w/v) lactose. B. subtilis was grown aerobically at 37³C in Luria^Bertani (LB) broth [17]. 2.2. Preparation of cell-free extracts and band-shift assay Exponentially growing cells (50 ml) were harvested by centrifugation, suspended in 1 ml of bu¡er I (50 mM Tris pH 7.6; 1 mM DTT ; 0.1 mM PMSF ; 10% glycerol) and disrupted by sonication (4 cycles of 1 min at 14 000 Hz with 1-min interval with a Sonics apparatus (Vibra-cell) at 4³C. After centrifugation (20 min at 14 000Ug) at 4³C, extracts were dialyzed in bu¡er I and concentrated in a Centricon centrifugal ¢lter device (Amicon; molecular mass cut-o¡ 10 000) to a ¢nal concentration of 20 mg ml31 . Various amounts of extracts were mixed with 20 ng of end-labelled DNA fragment (see below), 1 Wg of salmon sperm DNA (Sigma) and bu¡er I to a ¢nal volume of 20 Wl. After 10 min incubation at 30³C samples were fractionated on 10% polyacrylamide gels [8] at 200 V for 1.5 h. Gels were vacuum-dried and subjected to radioautography. The intensity of the retarded and unretarded bands was analyzed densitometrically (Molecular Personal FX apparatus; Bio-Rad). 2.3. Preparation and labelling of DNA fragments Synthetic oligonucleotides (Gibco BRL, sequences are reported in Fig. 2A) were suspended in 1UTE (10 mM Tris^HCl pH 8.0; 0.1 mM EDTA) at 100 mM. Complementary oligonucleotides were mixed together at a ¢nal
concentration of 10 ng Wl31 and sequentially incubated at 88³C for 2 min, 65³C for 10 min, 37³C for 10 min and 22³C for 10 min to allow the annealing reaction. Double stranded DNA fragments (Oligo 1, 2 and 3) were end-labelled with [Q-32 P]ATP by the use of the Ready-To-Go T4 Polynucleotide Kinase kit (Pharmacia Biotech) according to the manufacturer's instructions. 2.4. Western blot analysis Cell-free crude extracts (40 Wg) were fractionated through 10% SDS^PAGE and the polypeptides electrotransferred to PVDF membranes (Bio-Rad) by a TransBlot-Cell apparatus (Bio-Rad) for 45 min at 420 mA (31 V). Membranes were quickly stained with Ponceau S (Sigma) to check polypeptide transfer, de-stained and saturated overnight at 4³C in 1UPBS pH 7.2 (8 mM Na2 HPO4; 2 mM NaH2 PO4; 10 mM NaCl); 0.5% Tween 20; 5% skim milk (powder). Membranes were then incubated with anti-HrcA antibodies raised against the HrcA protein of B. subtilis and successively with peroxidase-conjugated secondary antibodies (anti-rabbit; Sigma). Peroxidase-conjugated antibodies were visualized by the ECL method (Amersham) according to the manufacturer's instructions. 2.5. Gel-¢ltration and anion-exchange chromatography 2^5 mg of cell-free crude extract were loaded on a gel¢ltration SE/100/17 column (15 ml volume ; Bio-Rad). The elution bu¡er was 50 mM Tris pH 7.6; 50 mM NaCl, the £ow applied was 1 ml min31 and 1-ml fractions were collected. All fractions were concentrated in a Centricon centrifugal ¢lter device (Amicon ; cut-o¡ 10 000) to a ¢nal protein concentration of about 2^3 mg ml31 and tested by band-shift assay. The most active fraction was loaded onto an anion-exchange column (UNO Q1 1.3 ml volume ; Bio-Rad). The elution bu¡ers were: (A) 25 mM Tris pH 7.6; (B) 25 mM Tris pH 7.6; 0.5 M NaCl and the elution program as follows : up to 1.8 ml = bu¡er A; from 1.8 to 2.3 ml = sample loaded; from 2.3 to 15.3 ml = gradient from 0 to 50% bu¡er B; from 15.3 to 18.3 ml = bu¡er B; from 18.3 to 26.3 ml = bu¡er A. The £ow applied was 4 ml min31 and 1-ml fractions were collected. All fractions were dialyzed, concentrated and tested by band-shift assay as above. HrcA of S. thermophilus was eluted at an NaCl concentration of 244 mM. To estimate HrcA molecular mass, the most active CIRCE-binding fraction from the anion-exchange chromatography was loaded on a SUPERDEX G-75 2.5 ml column (Pharmacia Biotech SMART system) and eluted with 50 mM Tris pH 7.6, 50 mM NaCl bu¡er (£ow 50 Wl min31 ) with the following molecular mass markers : transferrin (77 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). All fractions were tested by band-shift assay.
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2.6. Denaturation and GroEL-dependent renaturation of HrcA HrcA puri¢ed by gel ¢ltration and ion-exchange chromatography as described in Section 2.5 was either diluted 75-fold in 25 mM pH 7.6 Tris bu¡er at room temperature for 18 h, or incubated at 37³C for 10 min in the presence of 2 M guanidine^HCl/1 mM DTT, causing in both cases a decrease of DNA-binding activity of about 80%. Diluted and guanidine-denatured HrcA samples were, respectively, concentrated and dialyzed (Amicon, 10 000 cut-o¡). 0.75 Wg of each sample was incubated at 37³C for 1 h in the presence of 0.5 mg ml31 GroEL (from E. coli, StressGene) and 10% glycerol. ATP (1 mM) and MgCl2 (10 mM) were then added and incubation at 37³C continued for 1 h. ATP was removed by dialysis as above, and the e¤ciency of renaturation tested by band-shift assay. GroEL-mediated protection of HrcA was analyzed incubating 0.75 Wg of undenatured HrcA (puri¢ed as above) at 50³C for 2 h in the absence and in the presence of GroEL (0.5 mg ml31 ), ATP and MgCl2 . Samples were dialyzed to remove ATP as described above and used in band-shift experiments. 3. Results and discussion 3.1. S. thermophilus has an HrcA-like protein In several bacterial species the expression of the heatshock operons dnaK and groESL is negatively controlled by the repressor protein HrcA [3]. To approach the study of stress response in S. thermophilus, we looked for the
Fig. 1. Western blot analysis. Identical amounts of S. thermophilus (lane 1) and B. subtilis (lane 2) crude extracts were fractionated by 12.5% polyacrylamide^SDS gel electrophoresis. Polypeptides were electro-transferred on to membranes, reacted with speci¢c anti-HrcA antibodies (raised against HrcA of B. subtilis) and visualized by the ECL method. Molecular mass markers (in kDa) are reported.
Fig. 2. A: Sequences of the CIRCE consensus element [3] and of Oligo 1, Oligo 2 and Oligo 3. Arrows, dashed arrows and boxed letters indicate the palindromic sequences, the interrupted palindromic sequence of Oligo 2 and the bases modi¢ed in Oligo 2 and 3, respectively. B: Bandshift experiment performed with 20 ng of end-labelled Oligo 1. 10 Wg of an S. thermophilus crude extract were reacted with labelled DNA in the absence (lane 1) and in the presence (lane 2) of a 400-fold excess of unlabelled Oligo 1, as speci¢c competitor. A control lane (lane 3) with only labelled DNA was also run. C: Band-shift experiments performed with 20 ng of end-labelled Oligo 1, Oligo 2 or Oligo 3 and 10 Wg of an S. thermophilus crude extract. Samples of the experiments of B and C were fractionated on a 10% polyacrylamide gel. For all three DNA fragments, control reactions with only labelled DNA were also run.
presence of an HrcA-like protein by Western blot analysis. Identical amounts of S. thermophilus and B. subtilis crude extracts (40 Wg of proteins) were gel-fractionated and probed with antibodies raised against HrcA of B. subtilis (a gift of W. Shumann, University of Bayreuth, Germany). As shown in Fig. 1, a polypeptide of about 40 kDa was speci¢cally recognized among all the S. thermophilus proteins, suggesting structural similarities with the HrcA proteins of B. subtilis. The latter protein showed an apparent molecular mass of about 34 kDa, that does not precisely correspond to the size of 39 kDa deduced from amino acid sequence analysis for B. subtilis HrcA [18]. The S. thermophilus protein showed, instead, an apparent molecular mass of about 40 kDa, which conforms well with the
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size expected by deduced amino acid sequences for HrcA of various organisms [4,18] and suggests that the di¡erence observed in Fig. 1 is probably due to an anomalous migration of the B. subtilis HrcA on SDS^PAGE. 3.2. S. thermophilus contains a protein that binds the CIRCE element HrcA interacts with the CIRCE element, a palindromic sequence present in the promoter region of the dnaK and groESL operons of several bacteria [3]. Based on the CIRCE consensus sequence [3], two 27-bp long, complementary oligonucleotides (Section 2 and Fig. 2A) were synthesized, annealed and end-labelled. The obtained double strand DNA fragment, Oligo 1 (Fig. 2A), was then used in band-shift experiments with various amounts of S. thermophilus crude extracts. As shown in Fig. 2B, the electrophoretic mobility of labelled Oligo 1 was reduced in the presence of 1 Wg of S. thermophilus total proteins. This suggested that a polypeptide present in the extracts was able to bind Oligo 1 thus reducing its electrophoretic mobility. A densitometric analysis revealed that in our experimental conditions, with 1 Wg of S. thermophilus protein, about 0.85% (0.17 ng) of the labelled Oligo 1 was retarded (Section 2.2). Two lines of evidence indicated that this interaction was speci¢c for the CIRCE consensus sequence contained in Oligo 1: (i) binding was completely abolished by addition of 400-fold excess of unlabelled Oligo 1 to the reaction mixture (Fig. 2B), but not by 1000-fold excess of unspeci¢c (salmon sperm) DNA (not shown) ; (ii) DNA frag-
ments carrying a CIRCE element with either a disrupted palindromic sequence (fragment Oligo 2, Fig. 2A) or a palindromic sequence with an altered primary structure (fragment Oligo 3, Fig. 2A), were not recognized by proteins present in the crude extract (Fig. 2C). 3.3. Characterization of the S. thermophilus CIRCE-binding protein An S. thermophilus cell-free extract was fractionated by gel-¢ltration chromatography and all the collected fractions were analyzed by band-shift assay with labelled Oligo 1. The proteins contained in the most active CIRCEbinding fraction were further separated by anion-exchange chromatography and all the collected fractions tested again by band-shift assay. The fraction with the highest CIRCE-binding activity was analyzed by SDS^PAGE and showed a single polypeptide after Coomassie blue staining of the gel (data not shown). The single polypeptide of 40 kDa apparent molecular mass was speci¢cally recognized by anti-HrcA antibodies (data not shown) and conformed well with the size of the polypeptide visualized in the Western blot of Fig. 1. The chromatographic fraction yielding the single band was loaded on a gel ¢ltration chromatography (SMART) system (Pharmacia Biotech) with a Superdex G-75 column to estimate its native molecular mass. A single fraction showed DNA-binding activity in band-shift experiments with labelled Oligo 1 (not shown). The elution volume of the only fraction with CIRCE-binding activity (1.04 ml), reported on a calibration curve, allowed us to calcu-
Fig. 3. GroEL-mediated recovery of DNA-binding activity of HrcA following denaturation of the latter either chemically (A) or by dilution (B). 0.75 Wg of denatured HrcA were renatured in the absence (lanes 1 in both panels) or in the presence (lanes 2 in both panels) of 6-fold excess of GroEL and incubated with 20 ng of end-labelled Oligo 1. Control reactions in which, in the absence of puri¢ed HrcA, 20 ng of end-labelled Oligo 1 were reacted with 4.5 Wg of puri¢ed GroEL or run alone are reported in panel A (lanes 3 and 4, respectively). Samples were fractionated on 10% polyacrylamide gels.
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not have any detectable e¡ect on CIRCE-binding activity (Fig. 4, lanes 1 and 2). Our results indicate, in agreement with previous reports [10,19], that GroEL is needed in vitro to restore DNAbinding activity of a partially denatured HrcA. Further experiments will be required to establish the in vivo role of GroEL on HrcA function. 3.5. GroEL protects HrcA from heat denaturation in vitro
Fig. 4. Protection of HrcA-binding activity from heat-denaturation. Puri¢ed HrcA was incubated at standard (lanes 1 and 2) and high (lanes 3 and 4) temperature in the absence (lanes 1 and 3) and in the presence (lanes 2 and 4) of a 6-fold excess of GroEL (Section 2.6). Band-shift reactions were then performed with 20 ng of end-labelled Oligo 1 and samples were fractionated on 10% polyacrylamide gel.
late 74.5 kDa as the native molecular mass of the S. thermophilus DNA-binding protein. Taken together our results indicate that S. thermophilus contains an HrcA protein with a native molecular mass of about 74.5 kDa composed of at least one subunit of about 40 kDa (Fig. 1). Although we can not rule out the possibility that HrcA is an heterodimer formed by two subunits of very similar molecular mass, the simplest interpretation of our results is that, in S. thermophilus, HrcA is only active in an homodimeric form, as previously suggested in the case of B. subtilis [10]. 3.4. Recovery of HrcA-binding activity depends on GroEL Mogk et al. [10] reported that Bacillus stearothermophilus HrcA puri¢ed from E. coli needs GroEL for its DNAbinding activity. More recently, Minder et al. [19] have shown that B. japonicum HrcA, also puri¢ed from E. coli, does not need GroEL for maximal activity. This difference was explained suggesting that B. stearothermophilus HrcA was puri¢ed in a partially denatured form, requiring GroEL to recover its activity [19]. In agreement with this interpretation, Fig. 3 shows that partially denatured HrcA (with about 20% of remaining activity (Section 2.6)) needs GroEL to recover DNA-binding activity. Addition of GroEL to undenatured HrcA did
S. thermophilus is a moderately thermophilic bacterium with an optimal growth temperature of 42³C. A temperature upshift to 50³C induces the heat-shock response, as estimated by the induction of the synthesis of a 60-kDa protein, having N-terminal amino acid residues homologous to those of L. lactis GroEL (R. Raniello and L. Martirani, unpublished data). S. thermophilus HrcA showed a partial decrease of its in vitro DNA-binding activity when incubated at 50³C for 2 h (Fig. 4, lanes 1 and 3). Addition of a molar excess (6:1) of GroEL from E. coli (StressGene) before temperature upshift strongly reduced HrcA heat denaturation (Fig. 4, lanes 2 and 4), indicating a protecting e¡ect of GroEL on HrcA. GroEL-induced restoration of activity of partially denatured HrcA (Fig. 3) and protection from heat denaturation (Fig. 4) may be the result of a general chaperone activity. However it is tempting to hypothesize that in S. thermophilus, as shown before for other Gram-positive bacteria [10], GroEL is involved in HrcA binding to the CIRCE regulatory element. Acknowledgements We thank Wolfgang Shumann for the generous gift of anti-HrcA antibodies and critical reading of the manuscript. This work was supported by EU contract no BIO4CT960439, by Italian National Research Council (`Valorizzazione dei prodotti alimentari tipici mediterranei' and PF Biotecnologie) and by Italian MURST (PRIN 1999) to M.D.F.
References [1] Gross, C.A. (1996) Function and regulation of the heat shock proteins. In: Escherichia coli and Salmonella : Cellular and Molecular Biology (Neidhardt F.C. Ed.), pp. 1382^1399. ASM, Washington, DC. [2] Georgellis, D., Sohlberg, F., Hartl, U. and von Gabain, A. (1995) Identi¢cation of GroEL as a constituent of an mRNA-protection complex in Escherichia coli. Mol. Microbiol. 16, 1259^1268. [3] Hecker, M., Shumann, W. and Volker, U. (1996) Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19, 417^ 428. [4] Kilstrup, M., Jacobsen, S., Hammer, K. and Vogensen, F.K. (1997)
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[5]
[6]
[7]
[8]
[9] [10]
[11] [12]
L. Martirani et al. / FEMS Microbiology Letters 198 (2001) 177^182 Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis. Appl. Environ. Microbiol. 63, 1826^1837. Homuth, G., Masuda, S., Mogk, A., Kobayashi, Y. and Shumann, W. (1997) The dnaK operon of Bacillus subtilis is heptacistronic. J. Bacteriol. 179, 1153^1164. Zuber, U. and Shumann, W. (1994) CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176, 1359^1363. Van Asseldonk, M., Simons, M., Visser, H., de Vos, W. and Simons, G. (1993) Cloning, nucleotide sequence and regulatory analysis of the Lactococcus lactis dnaJ gene. J. Bacteriol. 175, 1637^1644. Duchene, A.M., Thompson, C.J. and Mazodier, P. (1994) Transcriptional analysis of the groEL genes in Streptomyces coelicolor A3. Mol. Gen. Genet. 245, 61^68. Yura, T., Nagai, H. and Mori, H. (1993) Regulation of the heat shock response in bacteria. Annu. Rev. Microbiol. 47, 321^350. Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F.X. and Shumann, W. (1997) The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J. 16, 4579^4590. Rallu, F., Gruss, A. and Maguin, E. (1996) Lactococcus lactis and stress. Antonie van Leeuwenhoek 70, 243^251. Broadbent, J.R., Oberg, C.J. and Wei, L. (1998) Characterization of the Lactobacillus helveticus groESL operon. Res. Microbiol. 149, 247^253.
[13] Walker, C.D., Girgis, H.S. and Klaenhammer, T.R. (1999) The groESL operon of Lactobacillus johnsonii. Appl. Environ. Microbiol. 65, 3033^3041. [14] Mollet, B., Knol, J., Poolman, B., Marciset, O. and Delley, M. (1993) Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus. J. Bacteriol. 175, 4315^ 4324. [15] Youngman, P., Perkins, J.B. and Losick, R. (1984) A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjiacent to Tn917 insertion. Mol. Gen. Genet. 195, 424^433. [16] Terzaghi, B.E. and Sandine, W.E. (1975) Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807^813. [17] Miller, J.H. (1972), Experiments in Molecular Genetics, pp. 433, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [18] Wetzstein, M., Volker, U., Dedio, J., Lobau, S., Zuber, U., Schiesswohl, M., Herget, C., Hecker, M. and Shumann, W. (1992) Cloning, sequencing and molecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol. 174, 3300^3310. [19] Minder, A.C., Fischer, H.M., Hennecke, H. and Narberhaus, F. (2000) Role of HrcA and CIRCE in the heath shock regulatory network of Bradyrhizobium japonicum. J. Bacteriol. 182, 14^22.
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Identi¢cation of the DNA-binding protein, HrcA, of Streptococcus thermophilus Luca Martirani a , Ra¡aella Raniello a , Gino Naclerio b , Ezio Ricca a , Maurilio De Felice a; * a
Department of General and Environmental Physiology, Section of Microbiology, University Federico II, via Mezzocannone 16, 80134 Naples, Italy b Faculty of Science, University of Molise, Via Mazzini 8, 86170 Isernia, Italy Received 5 March 2001; received in revised form 15 March 2001; accepted 15 March 2001
Abstract HrcA is a negative transcriptional factor controlling the expression of the stress-specific operons dnaK and groESL in several bacteria. Although the HrcA structural gene has been identified in various organisms, studies at the protein level have been so far limited and mostly restricted to Bacillus subtilis. We have identified the HrcA protein of Streptococcus thermophilus and show here that it is a dimer with a native molecular mass of 74.5 kDa and a sequence-specific DNA-binding activity. Partially denatured and inactive S. thermophilus HrcA recovered its binding activity in the presence of the GroEL chaperone. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : HrcA; DNA-binding protein; Streptococcus thermophilus
1. Introduction In natural environments bacteria often face stressful conditions, like nutrient starvation, temperature-, pH-, oxygen- and osmotic-shocks, that negatively a¡ect cell growth and viability. Bacteria are able to adapt their metabolism to and protect their cellular structures from the stressful conditions with the action of speci¢c stress proteins. Among the stress proteins, particularly important are the products of the dnaK and groESL operons that form the DnaK-DnaJ-GrpE and GroEL-GroES chaperone machines, respectively. These two multiprotein complexes are involved in several cellular processes, like folding of nascent polypeptides, maintaining proteins to be secreted in a secretion-competent form and refolding of partially denatured proteins [1]. In Escherichia coli GroEL also acts as an RNA chaperone which protects mRNA from nucleases [2].
* Corresponding author. Tel. : +39 (81) 2534638; Fax: +39 (81) 5514437; E-mail: [email protected]
The dnaK and groESL operons are normally transcribed at a low basal level and are induced in response to heat shock or to other conditions that increase the cytoplasmic levels of denatured proteins [1,3,4]. Two main transcriptional control mechanisms have been so far described: (i) a positive one, involving the stress-speci¢c transcriptional factor c32 , extensively characterized in E. coli [1], and (ii) a negative one involving the repressor protein HrcA [3]. HrcA is a DNA-binding protein, encoded by hrcA, the promoter proximal gene of the dnaK operon in Bacillus subtilis [5]. The HrcA-binding site is the CIRCE (controlling inverted repeat of chaperone expression) element [6], a palindromic sequence present in the promoter region of the dnaK and groESL operons of several bacteria [3]. In addition, the CIRCE element has been found in the promoter region of the dnaJ gene of Lactococcus lactis [7] and the groESL operon of Streptomyces coelicolor, Streptomyces albus and Bradyrhizobium japonicum [8,9]. Mogk et al. [10] have proposed that in B. subtilis HrcA is kept in an active form by interaction with GroEL. During normal growth, GroEL would stabilize HrcA which, in turn, binds to CIRCE, thus blocking groESL and dnaK transcription. Upon stress induction, accumulation of unfolded proteins within the cell would sequester GroEL, which causes HrcA inactivation and, consequently, active transcription of groESL and dnaK.
0378-1097 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 4 2 - 2
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Here we report the identi¢cation and characterization of HrcA from Streptococcus thermophilus. This organism is a moderately thermophilic lactic acid bacterium (LAB) that, in spite of its extensive biotechnological utilization, has so far been the subject of only limited studies. The understanding of stress response mechanisms in S. thermophilus is of particular interest, since during industrial fermentations this organism is exposed to stress conditions such as media acidi¢cation, temperature changes, high osmotic pressure and changes in oxygen concentration. The presence of HrcA has previously been suggested for other LAB, like L. lactis [7,11], Lactobacillus helveticus [12] and Lactobacillus johnsonii [13], based only on nucleotide sequence homologies and this is to our knowledge the ¢rst report of a direct characterization of HrcA from a LAB. 2. Materials and methods 2.1. Bacterial strains and media S. thermophilus and B. subtilis strains were ST11 [14] and PY79 [15], respectively. S. thermophilus was grown anaerobically at 42³C in M17 medium [16] supplemented with 1% (w/v) lactose. B. subtilis was grown aerobically at 37³C in Luria^Bertani (LB) broth [17]. 2.2. Preparation of cell-free extracts and band-shift assay Exponentially growing cells (50 ml) were harvested by centrifugation, suspended in 1 ml of bu¡er I (50 mM Tris pH 7.6; 1 mM DTT ; 0.1 mM PMSF ; 10% glycerol) and disrupted by sonication (4 cycles of 1 min at 14 000 Hz with 1-min interval with a Sonics apparatus (Vibra-cell) at 4³C. After centrifugation (20 min at 14 000Ug) at 4³C, extracts were dialyzed in bu¡er I and concentrated in a Centricon centrifugal ¢lter device (Amicon; molecular mass cut-o¡ 10 000) to a ¢nal concentration of 20 mg ml31 . Various amounts of extracts were mixed with 20 ng of end-labelled DNA fragment (see below), 1 Wg of salmon sperm DNA (Sigma) and bu¡er I to a ¢nal volume of 20 Wl. After 10 min incubation at 30³C samples were fractionated on 10% polyacrylamide gels [8] at 200 V for 1.5 h. Gels were vacuum-dried and subjected to radioautography. The intensity of the retarded and unretarded bands was analyzed densitometrically (Molecular Personal FX apparatus; Bio-Rad). 2.3. Preparation and labelling of DNA fragments Synthetic oligonucleotides (Gibco BRL, sequences are reported in Fig. 2A) were suspended in 1UTE (10 mM Tris^HCl pH 8.0; 0.1 mM EDTA) at 100 mM. Complementary oligonucleotides were mixed together at a ¢nal
concentration of 10 ng Wl31 and sequentially incubated at 88³C for 2 min, 65³C for 10 min, 37³C for 10 min and 22³C for 10 min to allow the annealing reaction. Double stranded DNA fragments (Oligo 1, 2 and 3) were end-labelled with [Q-32 P]ATP by the use of the Ready-To-Go T4 Polynucleotide Kinase kit (Pharmacia Biotech) according to the manufacturer's instructions. 2.4. Western blot analysis Cell-free crude extracts (40 Wg) were fractionated through 10% SDS^PAGE and the polypeptides electrotransferred to PVDF membranes (Bio-Rad) by a TransBlot-Cell apparatus (Bio-Rad) for 45 min at 420 mA (31 V). Membranes were quickly stained with Ponceau S (Sigma) to check polypeptide transfer, de-stained and saturated overnight at 4³C in 1UPBS pH 7.2 (8 mM Na2 HPO4; 2 mM NaH2 PO4; 10 mM NaCl); 0.5% Tween 20; 5% skim milk (powder). Membranes were then incubated with anti-HrcA antibodies raised against the HrcA protein of B. subtilis and successively with peroxidase-conjugated secondary antibodies (anti-rabbit; Sigma). Peroxidase-conjugated antibodies were visualized by the ECL method (Amersham) according to the manufacturer's instructions. 2.5. Gel-¢ltration and anion-exchange chromatography 2^5 mg of cell-free crude extract were loaded on a gel¢ltration SE/100/17 column (15 ml volume ; Bio-Rad). The elution bu¡er was 50 mM Tris pH 7.6; 50 mM NaCl, the £ow applied was 1 ml min31 and 1-ml fractions were collected. All fractions were concentrated in a Centricon centrifugal ¢lter device (Amicon ; cut-o¡ 10 000) to a ¢nal protein concentration of about 2^3 mg ml31 and tested by band-shift assay. The most active fraction was loaded onto an anion-exchange column (UNO Q1 1.3 ml volume ; Bio-Rad). The elution bu¡ers were: (A) 25 mM Tris pH 7.6; (B) 25 mM Tris pH 7.6; 0.5 M NaCl and the elution program as follows : up to 1.8 ml = bu¡er A; from 1.8 to 2.3 ml = sample loaded; from 2.3 to 15.3 ml = gradient from 0 to 50% bu¡er B; from 15.3 to 18.3 ml = bu¡er B; from 18.3 to 26.3 ml = bu¡er A. The £ow applied was 4 ml min31 and 1-ml fractions were collected. All fractions were dialyzed, concentrated and tested by band-shift assay as above. HrcA of S. thermophilus was eluted at an NaCl concentration of 244 mM. To estimate HrcA molecular mass, the most active CIRCE-binding fraction from the anion-exchange chromatography was loaded on a SUPERDEX G-75 2.5 ml column (Pharmacia Biotech SMART system) and eluted with 50 mM Tris pH 7.6, 50 mM NaCl bu¡er (£ow 50 Wl min31 ) with the following molecular mass markers : transferrin (77 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). All fractions were tested by band-shift assay.
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2.6. Denaturation and GroEL-dependent renaturation of HrcA HrcA puri¢ed by gel ¢ltration and ion-exchange chromatography as described in Section 2.5 was either diluted 75-fold in 25 mM pH 7.6 Tris bu¡er at room temperature for 18 h, or incubated at 37³C for 10 min in the presence of 2 M guanidine^HCl/1 mM DTT, causing in both cases a decrease of DNA-binding activity of about 80%. Diluted and guanidine-denatured HrcA samples were, respectively, concentrated and dialyzed (Amicon, 10 000 cut-o¡). 0.75 Wg of each sample was incubated at 37³C for 1 h in the presence of 0.5 mg ml31 GroEL (from E. coli, StressGene) and 10% glycerol. ATP (1 mM) and MgCl2 (10 mM) were then added and incubation at 37³C continued for 1 h. ATP was removed by dialysis as above, and the e¤ciency of renaturation tested by band-shift assay. GroEL-mediated protection of HrcA was analyzed incubating 0.75 Wg of undenatured HrcA (puri¢ed as above) at 50³C for 2 h in the absence and in the presence of GroEL (0.5 mg ml31 ), ATP and MgCl2 . Samples were dialyzed to remove ATP as described above and used in band-shift experiments. 3. Results and discussion 3.1. S. thermophilus has an HrcA-like protein In several bacterial species the expression of the heatshock operons dnaK and groESL is negatively controlled by the repressor protein HrcA [3]. To approach the study of stress response in S. thermophilus, we looked for the
Fig. 1. Western blot analysis. Identical amounts of S. thermophilus (lane 1) and B. subtilis (lane 2) crude extracts were fractionated by 12.5% polyacrylamide^SDS gel electrophoresis. Polypeptides were electro-transferred on to membranes, reacted with speci¢c anti-HrcA antibodies (raised against HrcA of B. subtilis) and visualized by the ECL method. Molecular mass markers (in kDa) are reported.
Fig. 2. A: Sequences of the CIRCE consensus element [3] and of Oligo 1, Oligo 2 and Oligo 3. Arrows, dashed arrows and boxed letters indicate the palindromic sequences, the interrupted palindromic sequence of Oligo 2 and the bases modi¢ed in Oligo 2 and 3, respectively. B: Bandshift experiment performed with 20 ng of end-labelled Oligo 1. 10 Wg of an S. thermophilus crude extract were reacted with labelled DNA in the absence (lane 1) and in the presence (lane 2) of a 400-fold excess of unlabelled Oligo 1, as speci¢c competitor. A control lane (lane 3) with only labelled DNA was also run. C: Band-shift experiments performed with 20 ng of end-labelled Oligo 1, Oligo 2 or Oligo 3 and 10 Wg of an S. thermophilus crude extract. Samples of the experiments of B and C were fractionated on a 10% polyacrylamide gel. For all three DNA fragments, control reactions with only labelled DNA were also run.
presence of an HrcA-like protein by Western blot analysis. Identical amounts of S. thermophilus and B. subtilis crude extracts (40 Wg of proteins) were gel-fractionated and probed with antibodies raised against HrcA of B. subtilis (a gift of W. Shumann, University of Bayreuth, Germany). As shown in Fig. 1, a polypeptide of about 40 kDa was speci¢cally recognized among all the S. thermophilus proteins, suggesting structural similarities with the HrcA proteins of B. subtilis. The latter protein showed an apparent molecular mass of about 34 kDa, that does not precisely correspond to the size of 39 kDa deduced from amino acid sequence analysis for B. subtilis HrcA [18]. The S. thermophilus protein showed, instead, an apparent molecular mass of about 40 kDa, which conforms well with the
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size expected by deduced amino acid sequences for HrcA of various organisms [4,18] and suggests that the di¡erence observed in Fig. 1 is probably due to an anomalous migration of the B. subtilis HrcA on SDS^PAGE. 3.2. S. thermophilus contains a protein that binds the CIRCE element HrcA interacts with the CIRCE element, a palindromic sequence present in the promoter region of the dnaK and groESL operons of several bacteria [3]. Based on the CIRCE consensus sequence [3], two 27-bp long, complementary oligonucleotides (Section 2 and Fig. 2A) were synthesized, annealed and end-labelled. The obtained double strand DNA fragment, Oligo 1 (Fig. 2A), was then used in band-shift experiments with various amounts of S. thermophilus crude extracts. As shown in Fig. 2B, the electrophoretic mobility of labelled Oligo 1 was reduced in the presence of 1 Wg of S. thermophilus total proteins. This suggested that a polypeptide present in the extracts was able to bind Oligo 1 thus reducing its electrophoretic mobility. A densitometric analysis revealed that in our experimental conditions, with 1 Wg of S. thermophilus protein, about 0.85% (0.17 ng) of the labelled Oligo 1 was retarded (Section 2.2). Two lines of evidence indicated that this interaction was speci¢c for the CIRCE consensus sequence contained in Oligo 1: (i) binding was completely abolished by addition of 400-fold excess of unlabelled Oligo 1 to the reaction mixture (Fig. 2B), but not by 1000-fold excess of unspeci¢c (salmon sperm) DNA (not shown) ; (ii) DNA frag-
ments carrying a CIRCE element with either a disrupted palindromic sequence (fragment Oligo 2, Fig. 2A) or a palindromic sequence with an altered primary structure (fragment Oligo 3, Fig. 2A), were not recognized by proteins present in the crude extract (Fig. 2C). 3.3. Characterization of the S. thermophilus CIRCE-binding protein An S. thermophilus cell-free extract was fractionated by gel-¢ltration chromatography and all the collected fractions were analyzed by band-shift assay with labelled Oligo 1. The proteins contained in the most active CIRCEbinding fraction were further separated by anion-exchange chromatography and all the collected fractions tested again by band-shift assay. The fraction with the highest CIRCE-binding activity was analyzed by SDS^PAGE and showed a single polypeptide after Coomassie blue staining of the gel (data not shown). The single polypeptide of 40 kDa apparent molecular mass was speci¢cally recognized by anti-HrcA antibodies (data not shown) and conformed well with the size of the polypeptide visualized in the Western blot of Fig. 1. The chromatographic fraction yielding the single band was loaded on a gel ¢ltration chromatography (SMART) system (Pharmacia Biotech) with a Superdex G-75 column to estimate its native molecular mass. A single fraction showed DNA-binding activity in band-shift experiments with labelled Oligo 1 (not shown). The elution volume of the only fraction with CIRCE-binding activity (1.04 ml), reported on a calibration curve, allowed us to calcu-
Fig. 3. GroEL-mediated recovery of DNA-binding activity of HrcA following denaturation of the latter either chemically (A) or by dilution (B). 0.75 Wg of denatured HrcA were renatured in the absence (lanes 1 in both panels) or in the presence (lanes 2 in both panels) of 6-fold excess of GroEL and incubated with 20 ng of end-labelled Oligo 1. Control reactions in which, in the absence of puri¢ed HrcA, 20 ng of end-labelled Oligo 1 were reacted with 4.5 Wg of puri¢ed GroEL or run alone are reported in panel A (lanes 3 and 4, respectively). Samples were fractionated on 10% polyacrylamide gels.
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not have any detectable e¡ect on CIRCE-binding activity (Fig. 4, lanes 1 and 2). Our results indicate, in agreement with previous reports [10,19], that GroEL is needed in vitro to restore DNAbinding activity of a partially denatured HrcA. Further experiments will be required to establish the in vivo role of GroEL on HrcA function. 3.5. GroEL protects HrcA from heat denaturation in vitro
Fig. 4. Protection of HrcA-binding activity from heat-denaturation. Puri¢ed HrcA was incubated at standard (lanes 1 and 2) and high (lanes 3 and 4) temperature in the absence (lanes 1 and 3) and in the presence (lanes 2 and 4) of a 6-fold excess of GroEL (Section 2.6). Band-shift reactions were then performed with 20 ng of end-labelled Oligo 1 and samples were fractionated on 10% polyacrylamide gel.
late 74.5 kDa as the native molecular mass of the S. thermophilus DNA-binding protein. Taken together our results indicate that S. thermophilus contains an HrcA protein with a native molecular mass of about 74.5 kDa composed of at least one subunit of about 40 kDa (Fig. 1). Although we can not rule out the possibility that HrcA is an heterodimer formed by two subunits of very similar molecular mass, the simplest interpretation of our results is that, in S. thermophilus, HrcA is only active in an homodimeric form, as previously suggested in the case of B. subtilis [10]. 3.4. Recovery of HrcA-binding activity depends on GroEL Mogk et al. [10] reported that Bacillus stearothermophilus HrcA puri¢ed from E. coli needs GroEL for its DNAbinding activity. More recently, Minder et al. [19] have shown that B. japonicum HrcA, also puri¢ed from E. coli, does not need GroEL for maximal activity. This difference was explained suggesting that B. stearothermophilus HrcA was puri¢ed in a partially denatured form, requiring GroEL to recover its activity [19]. In agreement with this interpretation, Fig. 3 shows that partially denatured HrcA (with about 20% of remaining activity (Section 2.6)) needs GroEL to recover DNA-binding activity. Addition of GroEL to undenatured HrcA did
S. thermophilus is a moderately thermophilic bacterium with an optimal growth temperature of 42³C. A temperature upshift to 50³C induces the heat-shock response, as estimated by the induction of the synthesis of a 60-kDa protein, having N-terminal amino acid residues homologous to those of L. lactis GroEL (R. Raniello and L. Martirani, unpublished data). S. thermophilus HrcA showed a partial decrease of its in vitro DNA-binding activity when incubated at 50³C for 2 h (Fig. 4, lanes 1 and 3). Addition of a molar excess (6:1) of GroEL from E. coli (StressGene) before temperature upshift strongly reduced HrcA heat denaturation (Fig. 4, lanes 2 and 4), indicating a protecting e¡ect of GroEL on HrcA. GroEL-induced restoration of activity of partially denatured HrcA (Fig. 3) and protection from heat denaturation (Fig. 4) may be the result of a general chaperone activity. However it is tempting to hypothesize that in S. thermophilus, as shown before for other Gram-positive bacteria [10], GroEL is involved in HrcA binding to the CIRCE regulatory element. Acknowledgements We thank Wolfgang Shumann for the generous gift of anti-HrcA antibodies and critical reading of the manuscript. This work was supported by EU contract no BIO4CT960439, by Italian National Research Council (`Valorizzazione dei prodotti alimentari tipici mediterranei' and PF Biotecnologie) and by Italian MURST (PRIN 1999) to M.D.F.
References [1] Gross, C.A. (1996) Function and regulation of the heat shock proteins. In: Escherichia coli and Salmonella : Cellular and Molecular Biology (Neidhardt F.C. Ed.), pp. 1382^1399. ASM, Washington, DC. [2] Georgellis, D., Sohlberg, F., Hartl, U. and von Gabain, A. (1995) Identi¢cation of GroEL as a constituent of an mRNA-protection complex in Escherichia coli. Mol. Microbiol. 16, 1259^1268. [3] Hecker, M., Shumann, W. and Volker, U. (1996) Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19, 417^ 428. [4] Kilstrup, M., Jacobsen, S., Hammer, K. and Vogensen, F.K. (1997)
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[5]
[6]
[7]
[8]
[9] [10]
[11] [12]
L. Martirani et al. / FEMS Microbiology Letters 198 (2001) 177^182 Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis. Appl. Environ. Microbiol. 63, 1826^1837. Homuth, G., Masuda, S., Mogk, A., Kobayashi, Y. and Shumann, W. (1997) The dnaK operon of Bacillus subtilis is heptacistronic. J. Bacteriol. 179, 1153^1164. Zuber, U. and Shumann, W. (1994) CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176, 1359^1363. Van Asseldonk, M., Simons, M., Visser, H., de Vos, W. and Simons, G. (1993) Cloning, nucleotide sequence and regulatory analysis of the Lactococcus lactis dnaJ gene. J. Bacteriol. 175, 1637^1644. Duchene, A.M., Thompson, C.J. and Mazodier, P. (1994) Transcriptional analysis of the groEL genes in Streptomyces coelicolor A3. Mol. Gen. Genet. 245, 61^68. Yura, T., Nagai, H. and Mori, H. (1993) Regulation of the heat shock response in bacteria. Annu. Rev. Microbiol. 47, 321^350. Mogk, A., Homuth, G., Scholz, C., Kim, L., Schmid, F.X. and Shumann, W. (1997) The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. EMBO J. 16, 4579^4590. Rallu, F., Gruss, A. and Maguin, E. (1996) Lactococcus lactis and stress. Antonie van Leeuwenhoek 70, 243^251. Broadbent, J.R., Oberg, C.J. and Wei, L. (1998) Characterization of the Lactobacillus helveticus groESL operon. Res. Microbiol. 149, 247^253.
[13] Walker, C.D., Girgis, H.S. and Klaenhammer, T.R. (1999) The groESL operon of Lactobacillus johnsonii. Appl. Environ. Microbiol. 65, 3033^3041. [14] Mollet, B., Knol, J., Poolman, B., Marciset, O. and Delley, M. (1993) Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus. J. Bacteriol. 175, 4315^ 4324. [15] Youngman, P., Perkins, J.B. and Losick, R. (1984) A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjiacent to Tn917 insertion. Mol. Gen. Genet. 195, 424^433. [16] Terzaghi, B.E. and Sandine, W.E. (1975) Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807^813. [17] Miller, J.H. (1972), Experiments in Molecular Genetics, pp. 433, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [18] Wetzstein, M., Volker, U., Dedio, J., Lobau, S., Zuber, U., Schiesswohl, M., Herget, C., Hecker, M. and Shumann, W. (1992) Cloning, sequencing and molecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol. 174, 3300^3310. [19] Minder, A.C., Fischer, H.M., Hennecke, H. and Narberhaus, F. (2000) Role of HrcA and CIRCE in the heath shock regulatory network of Bradyrhizobium japonicum. J. Bacteriol. 182, 14^22.
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