The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Marco Ventura, John G. Kenny, Ziding Zhang, Gerald F. Fitzgerald and Douwe van Sinderen

Alimentary Pharmabiotic Centre and Department of Microbiology, Bioscience Institute, National University of Ireland, Western Road, Cork, Ireland

Correspondence
Douwe van Sinderen
d.vansinderen{at}ucc.ie


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.


Abbreviations: HAIR, HspR-associated inverted repeat; HTH, helix–turn–helix; IR, inverted repeat

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY837845, AY837846 and AY842855.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bifidobacteria constitute a large group of bacteria, which are found mainly in the mammalian gastrointestinal tract. Although bifidobacteria represent only 3–6 % of the adult faecal flora, their presence has been associated with beneficial health effects, such as prevention of diarrhoea, amelioration of lactose intolerance, and immunomodulation (Scardovi, 1984; Ventura et al., 2004a). These correlations have led to the widespread use of bifidobacteria as live components of health-promoting or probiotic foods (Ventura et al., 2004a). The incorporation of bifidobacteria in such products requires that they survive industrial food manufacturing processes, such as starter handling and storage (freeze-drying, freezing or spray-drying), while remaining viable during storage. This reinforces the need for robust bifidobacteria that must also survive passage through the upper part of the digestive tract, while being able to compete with resident intestinal flora, preferably colonize the digestive tract, and express specific genes under what probably are suboptimal growth conditions. However, bifidobacteria are subjected to stressful environmental challenges not only during industrial processes, but also under natural conditions where their ability to quickly respond to stress is essential for survival.

In order to resist aggravating environmental conditions such as heat, cold and osmotic shock, bifidobacteria, like other bacteria, are capable of synthesizing a particular set of proteins protecting the cell from damage caused by the accumulation of unfolded and/or misfolded proteins (Wickner et al., 1999). Several of these protective proteins act as molecular chaperones, such as GroEL (Hsp60), DnaK (Hsp70) and ClpB (Hsp100) (Wickner et al., 1999), playing key roles in several posttranslational events to prevent protein denaturation, aggregation and misfolding (Georgopoulus & Welch, 1993). Recently, the groEL and dnaK genes, which were shown to be induced upon heat stress, have been investigated in bifidobacteria (Ventura et al., 2004b, 2005a).

The Clp proteases represent the most extensively studied chaperones from both a mechanistic and functional point of view (Chastanet & Msadek, 2003; Schelin et al., 2002; Schirmer et al., 1996; Squires & Squires, 1992; Wawrzynow et al., 1996; Wickner et al., 1999). The Clp holoenzyme consists of two separate and functionally distinct subunits. The proteolytic activity is provided by the ClpP subunit, which constitutes the Clp core. Hexamers of Clp ATPase subunits are associated with the core, and are required in order to recognize, unfold and present substrate proteins to ClpP (Wickner et al., 1999). In prokaryotes ClpB is a member of the Clp ATPase protein family, which is subdivided into two distinct groups. The first group includes large proteins (approximately 83 kDa) with two ATP-binding domains (represented by ClpA, ClpB, ClpC, ClpD and ClpE), while the second group includes smaller proteins (21–22 kDa) with a single ATP-binding domain (ClpM, ClpN, ClpX and ClpY) (Derre et al., 1999; Schirmer et al., 1996). Each group is further subdivided according to specific signature sequence motifs and the lengths of the interdomain region separating the nucleotide-binding domain (Schirmer et al., 1996). The ClpB ATPase possesses a sequence highly similar to that of ClpA, but does not appear to form a proteolytic complex with the ClpP subunit (Gottesman et al., 1998). In some organisms, the functional cooperation between ClpB and DnaK, DnaJ and GrpE is reflected in their gene organization and/or coordinated expression. In both Thermus thermophilus and Mycoplasma capricolum the clpB, dnaK, dnaJ and grpE genes are transcribed as a single operon (Falah & Gupta, 1997), whereas in Streptomyces the unlinked clpB and dnaK genes belong to the same regulon (Grandvalet et al., 1999).

In Escherichia coli, the heat-shock response activates transcription of more than 40 genes, including dnaK, dnaJ, grpE, lon and the Clp protease-encoding genes through positive control by {sigma}32 (Georgopoulus & Welch, 1993). In contrast to E. coli, heat-shock genes are generally negatively regulated in Gram-positive bacteria (Narberhaus, 1999). One of the most conserved heat-shock repressor systems described to date, not only in Gram-positive but also in some Gram-negative bacteria, is the HrcA/CIRCE system (Schulz & Schumann, 1996; Yuan & Wong, 1995). In the case of the high G+C content Gram-positive bacteria relatively little is known about the regulatory mechanisms controlling stress-induced genes (Bucca et al., 2003, 2000, 1995, 1997; Engels et al., 2004; Gottesman et al., 1998; Servant & Mazodier, 2001). The HspR repressor/operator system has been shown to regulate the dnaK operon and the lon gene of Streptomyces coelicolor (Bucca et al., 2003, 2000, 1995, 1997), and the clpB gene of Streptomyces albus (Grandvalet et al., 1999). Systems analogous to HspR are also present in other bacteria, including Mycobacterium tuberculosis (Stewart et al., 2002), Helicobacter pylori (Spohn & Scarlato, 1999) and Bifidobacterium breve (Ventura et al., 2005a).

In this report the clpB homologue of B. breve UCC 2003 is described. The transcriptional induction of this gene upon exposure to stressful conditions was investigated, while the role of a HspR homologue in the regulation of clpB was explored, revealing the first evidence for a global control system in the genus Bifidobacterium.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
All bacterial strains used are described in Table 1. All Bifidobacterium strains were grown anaerobically in MRS (Difco) supplemented with 0·05 % (w/v) L-cysteine-HCl and incubated at 37 °C for 16 h. E. coli was grown aerobically on a rotary shaker (150 r.p.m.) at 37 °C in LB medium, or plated onto LB Agar plates. Where appropriate, antibiotics were used at the following concentrations: 100 µg ampicillin ml–1 and 25 µg kanamycin ml–1. IPTG was added to a final concentration of 1 mM to induce expression of HspR in E. coli M15 (see below).


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Table 1. Bacterial strain and origin

 
Plasmids and plasmid constructions.
The E. coli pQE-30 vector (Qiagen) was used for overproduction and purification of an N-terminally histidine-tagged bifidobacterial HspR protein (h-HspR). The hspR gene from B. breve UCC 2003 was amplified using primers HSP-uni (5'-CGCGGATCCGCGCGGTTAGCCAACCAG-3') and HSP-rev (5'-CCCAAGCTTTCACCAACCCCACAGGAC-3'), containing a BamHI and a HindIII restriction sites, respectively (underlined). The resultant 584 bp PCR fragment was digested with BamHI and HindIII restriction enzymes, ligated into the similarly restricted vector using T4 DNA ligase enzyme (Roche) to generate plasmid pQE-Hsp, which was verified, following introduction into E. coli M15 (Qiagen), by electroporation and sequence analysis of plasmid DNA from several transformants. All DNA manipulation procedures were performed as described by Sambrook & Russell (2001).

DNA amplification and cloning of the clpB gene.
PCR was used to amplify a clpB homologue from Bifidobacterium animalis subsp. animalis strain ATCC 25527. A DNA fragment corresponding to the complete clpB homologue was amplified using oligonucleotides CLP-UNI (5'-CTCCACCGAGCACCTGC-3') and CLP-REV (5'-GTGATGCGACGGTCGGT-3'). Each PCR mixture (50 µl) contained 20 mM Tris/HCl, 50 mM KCl, 200 µM each deoxynucleoside triphosphate, 50 pmol each primer, 1·5 mM MgCl2 and 1 U Taq DNA polymerase (Gibco-BRL). Each PCR cycling profile consisted of an initial denaturation step of 5 min at 95 °C, followed by amplification for 35 cycles as follows: denaturation (30 s at 95 °C), annealing (30 s at 51 °C) and extension (1 min at 72 °C). The PCR reaction was completed with an elongation step (10 min at 72 °C). The resulting amplicons were separated with a 0·8 % (w/v) agarose gel, which was then stained with ethidium bromide. PCR fragments were purified using a PCR purification spin kit (Qiagen) and sequenced. The surrounding regions of the clpB homologues from B. animalis subsp. animalis ATCC 25527 and Bifidobacterium suis JCM 1269 were determined by inverse PCR; 1 µg chromosomal DNA was digested with the restriction endonucleases EcoRV or HindIII, the restriction fragments were then self-ligated and amplified using the primer pair CLP-1-inv (5'-GATCAGTCGAGCTTGCCTTC-3') and CLP-2-inv (5'-GCAAGATCGTCGACCTGCAG-3') as described by Sambrook & Russell (2001). The inverse PCR products obtained were then employed as templates for sequencing using a primer walking strategy.

Overproduction of h-HspR in E. coli.
In order to achieve overproduction of the B. breve h-HspR protein a 300 ml culture of the E. coli M15 strain containing the pQE-HspR plasmid was grown until it reached OD600 0·6, at which point the protein was induced by the addition of 1 mM IPTG. Three hours after induction, cells were harvested by centrifugation at 10 000 r.p.m. for 10 min. Cell pellets were resuspended in lysis buffer (100 mM NaH2PO4, 10 mM Tris/HCl, 6 M guanidine hydrochloride, pH 8·0) as recommended by the supplier (Qiagen), and allowed to lyse by shaking gently at 27 °C for 2 h. Cell debris was eliminated from the lysate by centrifugation at 13 000 r.p.m. for 10 min. The resulting supernatant was passed through a column containing 4 ml Ni-NTA agarose (Qiagen), which had been pre-equilibrated with 10 ml lysis buffer. The column was washed twice with 10 ml wash buffer (100 mM NaH2PO4, 10 mM Tris/HCl, 8 M urea, pH 6·3), and then eluted using 10 ml elution buffer (100 mM NaH2PO4, 10 mM Tris/HCl, 8 M urea, pH 5·9). The eluted purified h-HspR was renatured by dialysis at 4 °C against renaturation buffers containing 25 mM Tris/HCl pH 7·5, 1 mM EDTA, 1 mM NaCl, 10 mM DTT, 25 % (v/v) glycerol and a decreasing amount of urea (6, 4, 2 and 0 M) upon stepwise dialysis change. Protein concentrations were determined using the Bio-Rad protein assay in conjunction with a BSA standard curve. The size and purity of the isolated HspR were verified by SDS-PAGE as described by Laemmli (1970) using a 4 % (w/v) stacking gel and a 12 % (w/v) separating gel. Protein sizes were compared to a prestained protein marker (New England Biolabs).

RNA isolation and Northern blot analysis.
B. breve UCC 2003 cells were grown at 37 °C to OD600 0·6. The temperature conditions and the time sampling were chosen in accordance to the optimal growth condition of B. breve UCC 2003, and to previous reports concerning stress response in bifidobacteria (Ventura et al., 2004, 2005a, b).

Heat stress was applied by transferring the culture to 43, 47 or 50 °C, while osmotic stress was applied by the addition of 5 M NaCl-containing prewarmed medium, to give a final NaCl concentration of 0·5 or 0·7 M. At various time points 30 ml culture was removed and briefly centrifuged to harvest the cells. Total RNA was isolated using the macaloid-acid extraction method (Ventura et al., 2003) and treated with DNase (Roche). RNA (15 µg) was electrophoresed on a 1·5 % agarose-formaldehyde denaturing gel, transferred to a Zeta-Probe blotting membrane (Bio-Rad) as described by Sambrook & Russell (2001), and fixed by UV cross-linking using a Stratalinker 1800 (Stratagene). PCR amplicons obtained with primers targeting the B. breve UCC 2003 clpB gene were labelled with [{alpha}-32P] using a random primed DNA labelling system (Roche), and purified with spin columns (Amersham). Hybridization steps were performed at 65 °C in 0·5 M NaHPO4 pH 7·2, 1·0 mM EDTA, 7·0 % (w/v) SDS. Following 18 h of hybridization, the membrane was rinsed twice for 30 min at 65 °C in 0·1 M NaHPO4 pH 7·2, 1·0 mM EDTA, 1 % (w/v) SDS, twice for 30 min at 65 °C in 0·1 mM NaHPO4 pH 7·2, 1·0 mM EDTA, 0·1 % (w/v) SDS, and exposed to Kodak Biomax MS Film (Eastman-Kodak).

Primer extension analysis.
The 5' end of the clpB-encompassing mRNA transcript was determined as described previously (Ventura et al., 2003) using the synthetic oligonucleotides clpB-prom1 (5'-CTGACGCAGCAACGCATC-3') or clpB-prom2 (5'-GACGCACTCGGCAGCGCGAC-3').

Gel shift DNA-binding assays.
A 250 bp DNA fragment corresponding to the clpB and dnaK promoter region was amplified by PCR with the primer pairs clp1 (5'-GTCTCGTCTTGAGGTTTC-3'), clp2 (5'-TGAGAGTGGTCAACCCCAA-3'), and dk-1 (5'-GAGTGGCCCGCGTGG-3'), dk-2 (5'-CTCCTTAATTATTCGTTTGTTC-3'), respectively. The resultant amplicon was purified using a spin-column (Amersham), and then end-labelled using [{gamma}-32P]dATP and T4 polynucleotide kinase (New England Biolabs). The level of radioactive labelling was measured using a Beckman LS multi-purpose scintillation counter (Fullerton).

Binding reactions were performed in a final volume of 20 µl containing the labelled probe and varying concentrations of protein in the presence of 1 µg calf thymus DNA in binding buffer (50 mM Tris/HCl pH 7·5, 50 mM NaCl, 10 mM MgCl2). Following incubation at 37 °C for 30 min, samples were loaded on a 4 % polyacrylamide gel and electrophoresed at 28 V cm–1 for 1 h. Bands were visualized by autoradiography at –70 °C using Kodak Biomax MS Film (Eastman-Kodak).

HspR-3D prediction.
The fold recognition of the HspR was performed with the aid of the protein structure prediction Meta server (http://bioinfo.pl/Meta/). Taking the crystal structure of 1r8d (resolution 2·7 Å) as the structure template, the sequence alignment between HspR and 1r8d (chain A and B) was generated using the profile–profile alignment algorithm (FFAS) (Rychlewski et al., 2000). Furthermore, the theoretical dimeric structure for HspR was built using the homology modelling package (WHAT IF) (Vriend, 1990).

Nucleotide sequence accession numbers.
The nucleotide sequence data of the clpB locus of B. breve UCC 2003 and B. animalis subsp. animalis ATCC 25527 were deposited in GenBank under accession nos AY837845 and AY837846, respectively. The sequence of the promoter region of B. suis LMG 21814 was deposited in GenBank under accession no. AY842855.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the clpB locus in B. breve
The highly conserved amino acid sequences of the ClpB proteins in Bifidobacterium longum and S. coelicolor were used to identify putative clpB genes in the B. breve UCC 2003 genome. In this way a single ORF was identified whose deduced protein product displays a high degree of similarity to predicted and experimentally proven ClpB proteins from a wide variety of organisms. The structural organization and location of the clpB locus in the chromosome of B. breve UCC 2003 and other bacteria are schematically displayed in Fig. 1. This comparative analysis showed that the most (97 %) similar protein to the predicted B. breve ClpB chaperone was the putative ClpB protein from B. longum (Schell et al., 2002), while homology levels of >=48 % were still observed between the predicted B. breve ClpB protein and the ClpB protein of the unrelated bacterial taxum Clostridium perfringens. At the DNA level, significant sequence homology was still detectable between the various clpB genes, in contrast to the flanking DNA regions, which were shown to be highly variable. The presumed B. breve UCC 2003 clpB gene is located directly downstream of a gene encoding a predicted permease and upstream of a gene (gltX2) whose deduced product shows significant similarity to a glutamyl tRNA synthetase.



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Fig. 1. (a) Schematic comparative representation of the clpB loci of B. breve UCC 2003 and various other bacteria. Each arrow indicates an ORF, the size of which is proportional to the length of the arrow. Predicted protein function is indicated above each arrow. Gene products sharing >=55 % amino acid similarity are linked by light blue shading, and those sharing <=54 % amino acid similarity are linked by light violet shading. The amino acid identity in percent is indicated. (b) Alignment of amino acid sequences for ClpB proteins from high G+C content Gram-positive bacteria with the sequence of E. coli ClpB. The regions containing the two ATP-binding domains (ATP-1 and ATP-2) are boxed. B, B. longum NCC2705 (Q8G4X4); Bbr, B. breve UCC2003; Myc, Mycobacterium bovis (AAD00218; Cor, Corynebacterium efficiens (Q8FM94); Stc, S. coelicolor (NP_733613); Sta, S. avermitilis; Ec, E. coli (NC_000913).

 
The overall gene organization surrounding clpB is not conserved among bifidobacteria. In fact, the comparison of the clpB chromosomal region of the two closely related B. breve and B. longum species showed differences in their gene inventory (Fig. 1a). This finding was also confirmed through the analysis of the clpB locus in a phylogenetic distant taxon, B. animalis subsp. animalis ATCC 25527. In this micro-organism the clpB gene is located directly downstream of the faaH gene (encoding a putative fumarylacetoacetate hydrolase) and upstream of a gene encoding a predicted copper-transporting ATPase (Fig. 1a).

The amino acid alignment with characterized prokaryotic ClpB ATPases showed that the presumptive ClpB of B. breve UCC 2003 possesses two nucleotide-binding regions, ATP-binding 1 (amino acids 180–413) and ATP-binding 2 (amino acids 545–719), harbouring the ATPase A and B boxes characteristic for the ClpB protein family (Fig. 1b). The two ATP-binding domains are separated by a spacer region of 130 amino acids (amino acids 414–544), and enclosed between a leader sequence of 179 amino acids at the N-terminus and a trailer sequence of 75 amino acids at the C-terminus.

Heat and osmotic induction of the B. breve UCC 2003 clpB gene
Transcription of many stress genes is known to be induced by heat or osmotic shock (van de Guchte et al., 2002; Ventura et al., 2004b, 2005a, b). In order to determine whether the B. breve UCC 2003 clpB gene is induced following heat or osmotic shock a slot-blot hybridization procedure was employed. The mRNA used in these experiments was isolated from B. breve UCC 2003 cultures grown for different lengths of time at temperatures ranging from 37 to 50 °C (Fig. 2a), or NaCl concentrations ranging from 0·5 to 0·7 M (Fig. 2b). Based on the intensity of the hybridization signal, the highest expression level of the clpB gene was shown to occur following exposure to 50 °C for 25 min, or incubation for 50 min in a medium containing 0·7 M NaCl (Fig. 2a, b), conditions that increased clpB mRNA levels by approximately eighteen- and twenty-fold, respectively (Fig. 2c, d).



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Fig. 2. Heat- (a) and osmotic-shock (b) induction of the B. breve UCC 2003 clpB gene. Total RNA (25 µg per slot) was isolated from B. breve UCC 2003 cells exposed for up to 150 min to a range of temperatures from 37 to 50 °C, and to 0·5–0·7 M NaCl, and was probed with 32P-labelled clpB. (c, d) The resulting hybridization signals obtained in the autoradiograms were quantified. The mRNA synthesized at 43, 47 and 50 °C, and at 0·5 and 0·7 M NaCl was normalized to the amount present at 37 °C. The time of heat- and osmotic-shock exposure is indicated and represented by different shading. (e) Schematic diagram of the B. breve UCC 2003 clpB locus. Hairpin symbols indicate predicted secondary structures. The identified transcript is depicted as a solid arrow, which points towards the 3'-end of the mRNA. (f) Northern blot analysis of B. breve UCC 2003 clpB transcription. RNA was isolated from a culture at the beginning of the experiment (lane 1), at 50 min after temperature shift to 43 °C (lane 2), at 50 min after temperature shift to 47 °C (lane 3), grown in the presence of NaCl at a concentration of 0·5 M for 50 min (lane 4), grown in the presence of NaCl at a concentration of 0·7 M for 50 min (lane 5), at 50 min upon temperature shift at 50 °C (lane 6), at 150 min upon temperature shift at 50 °C (lane 7), and grown in the presence of NaCl at a concentration of 0·7 M for 150 min (lane 8). The molecular masses calculated for the hybridization signal, and the position of 16S and 23S rRNA are indicated.

 
Characterization of clpB transcription activity by Northern blotting
The transcriptional regulation of clpB was analysed by Northern blotting using the clpB sequence as a probe. Total mRNA was isolated from B. breve UCC 2003 grown at 37 °C, following heat shock at 43, 47 or 50 °C, or following osmotic shock at 0·5 or 0·7 M NaCl. Northern blotting revealed a major hybridization signal corresponding to a 2·6 kb mRNA (Fig. 2e, f), which is the expected size of a monocistronic transcript that would only encompass the clpB gene. No clpB mRNA was detected at 37 °C, whereas clpB transcription was transiently induced by heat shock and osmotic shock, with maximum mRNA levels being observed about 50 min after the NaCl concentration had been increased to 0·7 M. Analysis of the nucleotide sequence of the clpB locus revealed that the gene was flanked at its 3' end by two inverted repeats [{Delta}G –13·6 and –12·2 kcals (–56·9 and 51·0 kJ, respectively)] that may function as rho-independent transcriptional terminator structures (Fig. 2e).

Identification of the transcription start site of the clpB gene
To determine the transcriptional start point of the clpB gene, primer extension analysis was performed using RNA extracted from B. breve cells grown in the presence of 0·7 M NaCl (Fig. 3a). An extension product was identified 80 nucleotides 5' to the predicted translational start site of the clpB gene (Fig. 3b). A weaker extension product of identical size was obtained using mRNA extracted from cultures grown at 50 °C (Fig. 3a). The result was confirmed using a second primer, clpB-prom2 (data not shown). Analysis of the putative promoter region of the clpB revealed a potential promoter-like sequence resembling the consensus –10 and –35 hexamers.



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Fig. 3. Determination of the B. breve UCC 2003 clpB transcription initiation site by primer-extension analysis. (a) Primer extension using the clpB-prom1 oligonucleotide. (b) Representation of the promoter region of the clpB gene. The deduced –10 hexamer is underlined, bold type with an asterisk denotes the transcription start point, the start codon is double underlined, and IR indicates putative regulatory sequences. (c) Comparison between the promoter regions of five clpB genes from different Actinobacteridae, which includes B. suis JCM 1269, B. longum NCC 2705, B. breve UCC 2003, B. animalis subsp. animalis ATCC 25527 and S. albus J1074 (AF076980).

 
The predicted promoter regions of clpB genes from two bifidobacterial strains (B. breve UCC 2003 and B. longum NCC 2705) and one Streptomyces strain were aligned in an attempt to identify putative regulatory elements (Fig. 3c). For completeness, we determined by inverse PCR the putative promoter region of the clpB gene from a closely related Bifidobacterium species (B. suis) and a more distantly related one (B. animalis subsp. animalis). The alignment of these promoter sequences revealed that the putative –10 box as well as the –35 box were reasonably well conserved in all bifidobacterial sequences. Interestingly, the adenine corresponding to the experimentally determined transcriptional start site is conserved among the bifidobacterial species, while an inverted repeat (IR) sequence was conserved among all four bifidobacteria examined (Fig. 3c). The sequence of this IR is identical to a regulatory structure termed HAIR (HspR-associated inverted repeat; TTGAGT N7 ACTCAA), which has been demonstrated to be recognized by the heat-shock protein regulator (HspR) in several high G+C content Gram-positive bacteria (Bucca et al., 1997; Grandvalet et al., 1999). Screening of the available bifidobacterial genome sequences, B. longum NCC 2705, the unpublished B. breve UCC 2003 and the unfinished B. longum DJ01A0, for these IRs demonstrated that it is present within the predicted promoter regions of the clpB and dnaK homologues of these three bifidobacteria (Fig. 4). However, a less extensive IR sequence (TGAG N9 CTCA) was identified in the putative promoter region of 16 genes (Fig. 4). These include genes like mutT, trmD and malQ1, which are predicted to encode MutT, a methyltransferase and a glucanotransferase, respectively. Transcription of mutT, trmD and malQ1 homologues in other organisms has been shown to be induced following osmotic, heat or oxidative stress in high and low G+C content bacteria (Chamnongpol & Groisman, 2002; Frees et al., 2001; Taddei et al., 1997; Wolf et al., 2003). Moreover, IR-like sequences were identified upstream of bifidobacterial genes whose products were similar to a magnesium transporter (ORF a), a permease of drug metabolites (ORF b), a deoxyxylulose synthase (ORF c), a heat-responsive protein (ORF d), and two hypothetical proteins (ORF e and f). When we aligned the DNA sequences encompassing the IR motifs in all these putative promoter regions (Fig. 4), a putative consensus regulatory sequence could be identified, with the sequence aNaN2tTGAG N9 CTCAaN2tNtN2g.



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Fig. 4. IR sequence in different bifidobacterial sequences. Shading indicates conservation at a given position in at least 50 % of the nucleotides in the alignment as either identical (black) or similar (light grey) residues. The position of the sequence with respect to the predicted start codon is given. The last line indicates the consensus sequence.

 
An hspR homologue in B. breve UCC 2003
Analysis of the B. breve UCC 2003 genome revealed a gene highly (51 %) homologous to the hspR gene of S. coelicolor. Since HspR has been experimentally shown to be involved in the regulation of S. albus clpB (Grandvalet et al., 1999) and S. coelicolor dnaK (Bucca et al., 1997), the role of B. breve UCC 2003 HspR in the regulation of clpB expression was investigated. The putative B. breve UCC 2003 HspR protein consists of 195 amino acids with a molecular mass of 21·8 kDa. Furthermore, the protein contains a putative helix–turn–helix (HTH) DNA-binding motif close to the N-terminus, and a coiled-coil structure that extends from position 80 to 110 in the amino acid sequence. This structure is consistent with the putative HspR repressor binding to the IR motif as part of a multimeric complex.

The genome sequences of many other members of the Actinobacteridae group besides B. breve and S. coelicolor are available, and were analysed to assess the extent of conservation of the HspR-encoding gene. It was possible to identify hspR-like genes in all Actinobacteridae genomes (e-value<=10–10) at a conserved genomic location downstream of the dnaK gene (Ventura et al., 2005a). An alignment of the HspR proteins from representative members of the individual Actinobacteridae genera is shown in Fig. 5(a). Interestingly, the region corresponding to the HTH was found to be highly conserved (Fig. 5a).



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Fig. 5. Alignment of HspR proteins from several Actinobacteridae (a). The amino acid sequence of B. breve UCC 2003 (AY642911) (Bb) was aligned with those of HspR proteins from S. coelicolor A3 (AAB29454 (Sc), Mycobacterium tuberculosis CDC1551 (NP_334776) (Mt), Thermobifida fusca (ZP_00292291) (Tf), Corynebacterium glutamicum ATCC 1302 (NP_601989) (Cg), Nocardia farcinica IFM 10152 (YP_121622) (Nf), B. longum NCC 2705 (NP_695709) (Bl) and Propionibacterium acnes KPA171202 (YP_056708) (Pa). Shading indicates conservation at a given position in at least 50 % of the amino acid residues in the alignment as either identical (black) or similar (grey) residues. Dotted lines and bars below the aligned sequences indicate {alpha}-helices and turns, respectively, comprising the DNA-binding domain; HTH (residues 44–64) indicates the typical HTH motif described in Mer-like transcriptional regulators; W1 and W2 indicate wings 1 (residues 70–81) and 2 (residues 81–116). The amino acid residues involved in DNA binding are indicated. (b, c) The predicted 3D model of HspR. (b) The HspR dimer. One monomer is coloured violet; the other is coloured green. To provide a definition of the binding between HspR and the DNA molecule, the DNA molecule (the ligand) from the crystal structure of MtaN (1r8d) is superimposed into the predicted HspR model, and is indicated by grey tubes. (c) The potential residues in HspR involved in the interaction with the DNA molecule are indicated. The atoms in these highlighted residues are represented as sticks and are coloured as follows: blue, nitrogen; red, oxygen.

 
Structural investigation on HspR of B. breve UCC 2003
Using fold recognition prediction several structural homologues of HspR (Changela et al., 2003; Heldwein & Brennan, 2001; Newberry & Brennan, 2004) could be identified. These include the copper-efflux regulator of E. coli (CueR; PDB entry 1q05) (Changela et al., 2003), the transcription activator for the multidrug-transporter gene from Bacillus subtilis (BmrR; PDB entry 1exi) (Heldwein & Brennan, 2001), and the N-terminus of a global multidrug-transporter activator from Bacillus subtilis (MtaN; PDB entry 1r8d) (Newberry & Brennan, 2004). All these transcriptional regulators are dimeric proteins that contain a homologous N-terminal DNA-binding domain linked by a variable length coiled-coil to a C-terminal ligand-specific ‘coactivator’ binding domain. Although the overall protein sequence identity between the HspR of B. breve UCC 2003 and these proteins is only 17–21 %, the results from fold recognition revealed that the predicted structure of HspR is similar to these proteins in the region that includes the DNA-binding domain (amino acid residues 41 to 147 of HspR).

Using Bacillus subtilis 1r8d (chain A and B) a theoretical dimeric structure for HspR was built via homology modelling (Fig. 5b), indicating that this protein belongs to the superfamily of winged-helix proteins (Heldwein & Brennan, 2001). Members of this protein superfamily contain a DNA-binding domain represented by the HTH motif as well as two ‘wings’, W1 and W2, all of which are present in the B. breve HspR (Fig. 5a, b). Dimer formation of HspR molecules may be stabilized primarily by the formation of an anti-parallel coiled-coil between the two long helices as has been demonstrated for similar proteins (Heldwein & Brennan, 2001; Martirani et al., 2001) (Fig. 5b).

The DNA contacts in the Bacillus subtilis MtaN-DNA complex have been shown to be very similar to those seen in the BmrR-DNA complex, which implicates eight amino acid residues at specific positions in such protein–DNA interactions (Newberry & Brennan, 2004). The equivalent positions for these eight amino acid residues have been identified in HspR of B. breve UCC 2003 and are displayed in Fig. 5(c).

B. breve UCC 2003 HspR binds to the clpB and dnaK promoter regions
In S. coelicolor and in S. albus the HspR protein interacts with three HAIR motifs in the vicinity of the dnaK promoter (Bucca et al., 1995, 1997) and with a HAIR motif in the promoter region of the clpB gene (Grandvalet et al., 1999). Since one and two copies of the IRs highly similar to the HAIR motif were detected in the region upstream of the –35 box of the B. breve UCC 2003 clpB and dnaK promoters (Ventura et al., 2005a), respectively, we investigated whether HspR is capable of binding directly to the IR motif of the clpB promoter region by gel retardation assays. Early attempts to isolate overexpressed native HspR from B. breve UCC 2003 were hampered by its tendency to form insoluble aggregates in E. coli. Consequently the N-terminally histidine-tagged version of HspR was purified by immobilized metal affinity chromatography under denaturing conditions, and then refolded under renaturing conditions (Fig. 6a). The binding activity of this purified h-HspR protein to the clpB promoter region (clpBp) containing the IR motif was assessed in a gel-shift assay (Fig. 6b).



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Fig. 6. Detection of HspR binding to the clpB promoter of B. breve UCC 2003. (a) Overproduction and purification of h-HspR. SDS-PAGE analysis of crude extracts from E. coli M15 carrying pEQ-HspR without IPTG induction (lane 1), with IPTG induction and cell precipitation by centrifugation (lane 2), with IPTG induction without cell precipitation (lane 3), or purified and renatured h-HspR protein after immobilized metal affinity chromatography (lane 4). Molecular mass standards (Bio-Rad) were loaded in lanes MK. Gel retardation assays (b, c) were performed with a 250 bp clpB promoter fragment (b) and with a 200 bp dnaK promoter fragment (c) as the probe. Lanes 1 and 15, no protein extract; lane 2, 100 pmol h-HspR; lane 3, 50 pmol h-HspR plus 2 µg crude lysate from B. breve UCC 2003 cultures (cl-UCC2003) grown at 37 °C; lane 4, 100 pmol h-HspR plus 2 µg cl-UCC2003 grown at 37 °C; lane 5, 2 µg cl-UCC2003 grown at 37 °C; lane 6, 50 pmol h-HspR plus 2 µg cl-UCC2003 grown at 43 °C; lane 7, 100 pmol h-HspR plus 2 µg cl-UCC2003 grown at 43 °C; lane 8, 2 µg cl-UCC2003 grown at 43 °C; lane 9, 50 pmol h-HspR plus 2 µg cl-UCC2003 grown at 50 °C; lane 10, 100 pmol h-HspR plus 2 µg cl-UCC2003 grown at 50 °C; lane 11, 2 µg cl-UCC2003 grown at 50 °C; lane 12, 50 pmol h-HspR plus 2 µg cl-UCC2003 grown in a media with 0·7 M NaCl; lane 13, 100 pmol h-HspR plus 2 µg cl-UCC2003 grown in a media with 0·7 M NaCl; lane 14, 2 µg cl-UCC2003 grown in a media with 0·7 M NaCl.

 
No binding activity of the h-HspR protein was detected (Fig. 6b, lane 2) when the purified h-HspR protein on its own was used in the binding assay. In contrast, when 50 or 100 pmol of purified h-HspR protein was used in the presence of 2 µg of a crude extract from B. breve UCC 2003 cultures grown at 37 or 43 °C a clear displacement of the clpBp fragment was observed (Fig. 6b, lanes 3, 4, 6, 7). In contrast, when 50 or 100 pmol of purified h-HspR protein was used in the presence of 2 µg of a crude extract from B. breve UCC 2003 cultures exposed to 50 °C, or grown in the presence of 0·7 M NaCl in the medium, no DNA shift of the clpBp fragment was observed (Fig. 6b, lanes 9, 10, 12, 13).

Furthermore, we assayed the binding activity of the purified B. breve UCC 2003 h-HspR protein to the dnaK promoter region (dnaKp) containing the IR motif by performing DNA gel-shift experiments (Fig. 6c). Similarly to what was observed for the clpB promoter region, the purified h-HspR was not able to retard the dnaKp fragment when the purified h-HspR protein was employed alone (Fig. 6c, lane 2). However, when the purified h-HspR protein was incubated with 2 µg of a crude extract from B. breve UCC 2003 cultures grown at 37 or 43 °C a complete displacement of the dnaKp fragment was detected (Fig. 6c, lanes 3, 4, 6, 7). In contrast, the purified h-HspR protein incubated with crude extracts from B. breve UCC 2003 cultures grown at 50 °C or following osmotic shock at 0·7 M NaCl had no influence on the gel mobility of the dnaKp fragment (Fig. 6c, lanes 9, 10, 12, 13). In control experiments, 2 µg of a crude extract of the control strain grown at 37 or 43 °C, without h-HspR, did not affect the mobility of the clpBp fragment (Fig. 6b, lanes 5, 8) and the dnaKp fragment (Fig. 6c, lanes 5, 8).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Unlike the model organisms Bacillus subtilis and E. coli, relatively little is known about the regulatory mechanisms underlying stress response in high G+C content Gram-positive bacteria such as bifidobacteria. Analysis of the complete genome sequence of B. breve UCC 2003 and a number of recent reports (Schmidt & Zink, 2000; Ventura et al., 2004a, b, c, 2005a, b) indicate the existence of a variety of chaperone-encoding genes in bifidobacteria. This paper reports the characterization of a clpB homologue from bifidobacteria. The B. breve UCC 2003 clpB gene is transcriptionally induced as a monocistronic 2·6 kb transcript upon imposition of osmotic or severe heat stress.

The comparison of putative regulatory elements for the clpB gene from two bifidobacteria allowed the identification of a putative consensus promoter sequence (IR), which is highly similar to the HspR-binding site (HAIR) of S. coelicolor (Bucca et al., 1995, 1997, 2000, 2003) and S. albus (Grandvalet et al., 1999). Such HAIR sequences were shown to be involved in heat-shock regulation in Streptomyces, Mycobacterium and Helicobacter pylori (Grandvalet et al., 1999). It is worth noting that the HAIR motif of the S. albus clpB promoter (Grandvalet et al., 1999) is closely positioned to the transcriptional start site. In contrast, the IR motif of the promoter region B. breve clpB is placed immediately upstream to the putative –35 sequence, which may have a different effect on the strength of the repression.

The purified, renaturated, histidine-tagged HspR from B. breve UCC 2003 was not able to retard the clpBp and the dnaKp promoter fragments in gel shift experiments. However, a clear retardation was observed when crude lysate from non-stressed B. breve cultures was included in the binding assay. This indicates a requirement for one or more cofactors that work in concert with HspR to control clpB and dnaK gene expression in bifidobacteria. However, the nature and the mechanism by which these cofactors modulate the activity of HspR remain elusive. This finding is supported by the fact that HspR belongs to the MerR family, which includes regulatory proteins whose binding activity seems to be modulated by cofactors such as proteins or metal ions (Newberry & Brennan, 2004; Outten et al., 1999; Summers, 1992). Bucca et al. (2000) have previously suggested that cofactors join the dnaK regulation process in S. coelicolor, which would therefore also represent an example of activated HspR protein.

Interestingly, only the crude extract from UCC 2003 cultures grown under conditions where clpB and dnaK genes are not induced contains these cofactors that are involved in the clpB and dnaK regulatory process. Conversely, crude lysate from UCC 2003 cultures grown under conditions (e.g. exposure to severe heat or osmotic stresses) that activate clpB and dnaK gene transcription does not allow HspR to retard the mobility of clpBp and the dnaKp fragments. Therefore, these findings suggest that, similar to what has been described for other high G+C content bacteria (Narberhaus, 1999), HspR from B. breve UCC 2003 acts as a negative regulator of clpB and dnaK transcription, and this repressive action is relieved by one or more effector molecule(s). In contrast to the DnaK co-repressor model described for other members of the Actinobacteridae group (Bucca et al., 2000), in B. breve UCC 2003 the DnaK protein is unlikely to act as a cofactor of HspR. In fact, as clearly shown in a previous report (Ventura et al., 2005a), the B. breve UCC 2003 dnaK operon exhibits only marginal transcription under moderate shock regimes (37 and 43 °C), and thus the crude extract from B. breve UCC 2003 unstressed cells will be expected to contain only minimal amounts of DnaK protein. However, an alternative explanation for the observed results would be that the purified h-HspR is incorrectly folded and that the proper conformation is only achieved with the activity of a molecular chaperone contained in the crude extract from UCC 2003 cultures grown at 37 or 43 °C.

Recently it was observed that two IRs (TGAG N9 CTCA), which are highly similar to the HAIR consensus motif (Bucca et al., 2003, 2000, 1997, 1995; Grandvalet et al., 1999), are present in the promoter regions of four genes of B. longum NCC 2705 (Schell et al., 2002; Ventura et al., 2005a). These include classical stress-induced genes such as clpB and dnaK, which have been described as being associated with the stress response. The screening of the unpublished B. breve UCC 2003 genome and the unfinished B. longum DJO1A0 genome enriched the list of promoter regions that possess the IR sequence; these promoters may represent genes and operons that belong to the HspR regulon. These genes include those that are significantly homologous to genes with demonstrated involvement in the osmotic stress response (e.g. trmD, malQ, ORF c) (Frees et al., 2001; Wolf et al., 2003), heat stress response (ORF d) or general stress response (ORF a and mutT) (Chamnongpol & Groisman, 2002; Taddei et al., 1997).

The prediction of HspR folding highlights the importance of eight amino acid residues in the B. breve UCC 2003 HspR protein, and compounds its functional relationship to MerR-like transcriptional regulators (Heldwein & Brennan, 2001; Newberry & Brennan, 2004). These amino acid residues are located within the HTH (His-54, Gln-56, Arg-59, Gln-60 and Tyr-61), as well as in both wings, W1 (Arg-77) and W2 (Gln-95 and Leu-101) (Fig. 5a, c). Moreover, the Tyr-61, Arg-77 and Leu-101 residues are highly conserved among the MerR protein family members, thus implying their functional importance in DNA binding. In the MerR protein family members the homologous Asp-62 position of HspR is occupied by either an aspartate, glutamate or glutamine residue. The Asp-62 residue interacts through a hydrogen bond with the Arg-77 residue, and consequently it stabilizes the interaction between Arg-77 and the phosphate groups of the DNA molecule.

In other members of the Actinobacteridae group it has been shown that the dnaK operon and the clpB gene belong to the same regulon (Bucca et al., 2003; Grandvalet et al., 1999). This also seems to be the case for B. breve UCC 2003, in which expression of the dnaK operon and clpB is induced by osmotic shock and severe heat stress, but not by moderate heat stress (this work; Ventura et al., 2005a; unpublished results), suggesting an overlap between the osmotic-shock and severe heat-shock regulons. Interestingly, in bifidobacteria clpB and dnaK represent the first chaperone-encoding genes to be strongly induced after exposure to very high temperature ({Delta}T 13 K). In contrast, we have observed that maximal transcription of heat-stress-induced genes such as groESL (Ventura et al., 2004b), clpC (Ventura et al., 2005b) and clpP (unpublished results) occurs upon moderate heat-shock regimes ({Delta}T of 6 K). Notably, transcription of the latter two genes is not induced by osmotic stress (Ventura et al., 2005b; unpublished results). Thus two separate regulatory pathways for coping with different levels of stresses are operating in bifidobacteria. The first pathway corresponds to the HspR regulon that protects cells from protein damage occurring when bifidobacteria are exposed to severe heat and osmotic shocks, and the second pathway (ClgR) becomes activated when bifidobacterial cells are exposed to moderate heat stress.

The topic of stress response in bifidobacteria is highly relevant to the food industry. Key aspects related to industrial applications, such as preparation of cells using freezing/drying technologies, and survival in products that present a hostile environment for bifidobacteria, make it essential to improve our knowledge of the mechanisms of osmotic and heat shock.

Thus a key objective for the future will be to examine the B. breve heat- and osmotic-shock responses more globally at the transcriptome level, and to investigate the level of integration and cross-talk between the different regulons, which includes genes controlled by HspR, ClgR and HrcA.


   ACKNOWLEDGEMENTS
 
This work was financially supported by Enterprise Ireland (grant BR/1998/202), the Higher Education Authority Programme for Research in Third Level Institutions, the Science Foundation Ireland Alimentary Pharmabiotic Centre at the National University of Ireland, and a Marie Curie Development Host Fellowship (HPMD-2000-00027) to M. V. We would like to thank: S. Leahy, J. A. Moreno-Munoz, M. O'Connell-Motherway and D. Higgins for providing the B. breve UCC 2003 genome sequences.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bucca, G., Ferina, G., Puglia, A. M. & Smith, C. P. (1995). The dnaK operon of Streptomyces coelicolor encodes a novel heat-shock protein which binds to the promoter region of the operon. Mol Microbiol 17, 663–674.[CrossRef][Medline]

Bucca, G., Hindle, Z. & Smith, C. P. (1997). Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor. J Bacteriol 179, 5999–6004.[Abstract/Free Full Text]

Bucca, G., Brassington, A. M. E., Schonfeld, H. J. & Smith, C. P. (2000). The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressor. Mol Microbiol 38, 1093–1103.[CrossRef][Medline]

Bucca, G., Brassington, A. M. E., Hotchkiss, G., Mersinias, V. & Smith, C. P. (2003). Negative feedback regulation of dnaK, clpB and lon expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis. Mol Microbiol 50, 153–166.[CrossRef][Medline]

Chamnongpol, S. & Groisman, E. A. (2002). Mg2+ homeostasis and avoidance of metal toxicity. Mol Microbiol 44, 561–571.[CrossRef][Medline]

Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., O'Halloran, T. V. & Mondragon, A. (2003). Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387.[Abstract/Free Full Text]

Chastanet, A. & Msadek, T. (2003). clpP of Streptococcus salivarius is a novel member of the dually regulated class of stress response genes in Gram-positive bacteria. J Bacteriol 185, 683–687.[Abstract/Free Full Text]

Derre, I., Rapoport, G. & Msadek, T. (1999). CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram-positive bacteria. Mol Microbiol 31, 117–131.[CrossRef][Medline]

Engels, S., Schweitzer, J.-E., Ludwig, C., Bott, M. & Schaffer, S. (2004). clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor {sigma}H. Mol Microbiol 52, 285–302.[CrossRef][Medline]

Falah, M. & Gupta, R. (1997). Phylogenetic analysis of Mycoplasma based on Hsp70 sequences: cloning of the dnaK (hsp70) gene of Mycoplasma capricolum. Int J Syst Bacteriol 47, 38–45.[Abstract/Free Full Text]

Frees, D., Varmanen, P. & Igmer, H. (2001). Stress tolerance and proteolysis in Lactococcus lactis. Mol Microbiol 41, 93–103.[CrossRef][Medline]

Georgopoulus, C. & Welch, W. J. (1993). Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 9, 601–634.[CrossRef][Medline]

Gottesman, S., Roche, E., Zhou, Y. N. & Sauer, R. T. (1998). The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev 12, 1338–1347.[Abstract/Free Full Text]

Grandvalet, C., Crécy-Lagard, V. & Mazodier, P. (1999). The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon. Mol Microbiol 31, 521–532.[CrossRef][Medline]

Heldwein, E. E. & Brennan, R. G. (2001). Crystal structure of the transcription activator BmrR bound to DNA and to a drug. Nature 409, 378–382.[CrossRef][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Martirani, L., Raniello, R., Naclerio, G., Ricca, E. & De Felice, M. (2001). Identification of the DNA-binding protein, HrcA of Streptococcus thermophilus. FEMS Microbiol Lett 198, 177–182.[CrossRef][Medline]

Narberhaus, F. (1999). Negative regulation of bacterial heat shock genes. Mol Microbiol 31, 1–8.[CrossRef][Medline]

Newberry, K. J. & Brennan, R. G. (2004). The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. J Biol Chem 279, 20356–20362.[Abstract/Free Full Text]

Outten, C. E., Outten, F. W. & O'Halloran, T. V. (1999). DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in Escherichia coli. J Biol Chem 274, 37517–37524.[Abstract/Free Full Text]

Rychlewski, L., Jaroszewski, L., Li, W. & Godzik, A. (2000). Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Sci 9, 232–241.[Abstract]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Scardovi, V. (1984). Genus Bifidobacterium Orla-Jensen, 1924, 472. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 1418–1434. Edited by N. R. Krieg & J. G. Holt. Baltimore, MD: Williams & Wilkins.

Schelin, J., Lindmark, F. & Clarke, A. K. (2002). The clp multigene family for the ATP-dependent Clp protease in the cyanobacterium Synechococcus. Microbiology 148, 2255–2265.[Medline]

Schell, M. A., Karmirantzou, M., Snel, B. & 9 other authors (2002). The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci U S A 99, 14422–14427.[Abstract/Free Full Text]

Schirmer, E. C., Glover, J. R., Singer, M. A. & Lindquist, S. (1996). HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem Sci 21, 289–296.[CrossRef][Medline]

Schmidt, G. & Zink, R. (2000). Basic features of stress response in three species of bifidobacteria: B. longum, B. adolescentis, and B. breve. Int J Food Microbiol 55, 41–45.[CrossRef][Medline]

Schulz, A. & Schumann, W. (1996). hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J Bacteriol 178, 1088–1093.[Abstract/Free Full Text]

Servant, P. & Mazodier, P. (2001). Negative regulation of the heat shock response in Streptomyces. Arch Microbiol 176, 237–242.[CrossRef][Medline]

Spohn, G. & Scarlato, V. (1999). The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori. Mol Microbiol 34, 663–674.[CrossRef][Medline]

Squires, C. & Squires, C. L. (1992). The Clp proteins: proteolysis regulators or molecular chaperones? J Bacteriol 174, 1081–1085.[Medline]

Stewart, G. R., Wernisch, L., Stabler, R., Mangan, J. A., Hinds, J. & Laing, K. G. (2002). Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148, 3129–3138.[Medline]

Summers, A. O. (1992). Untwist and shout: a heavy metal-responsive transcriptional regulator. J Bacteriol 174, 3097–3101.[Medline]

Taddei, F., Hayakawa, H., Bouton, M. F., Cirinesi, A. M., Matic, L., Sekiguchi, M. & Radman, M. (1997). Counteraction by MutT protein of transcriptional errors caused by oxidative damage. Science 278, 128–130.[Abstract/Free Full Text]

van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S. D. & Manguin, E. (2002). Stress responses in lactic acid bacteria. Antonie van Leeuwenhoek 82, 187–216.[CrossRef][Medline]

Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R. & Zink, R. (2003). Analysis, characterization and loci of the tuf genes in Lactobacillus and Bifidobacterium and their direct application for species identification. Appl Environ Microbiol 69, 6908–6922.[Abstract/Free Full Text]

Ventura, M., van Sinderen, D., Fitzgerald, G. F. & Zink, R. (2004a). Insights into the taxonomy, genetics and physiology of bifidobacteria. Antonie van Leeuwenhoek 86, 205–223.[CrossRef][Medline]

Ventura, M., Canchaya, C., Zink, R., Fitzgerald, G. F. & van Sinderen, D. (2004b). Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional and phylogenetic analysis. Appl Environ Microbiol 70, 6197–6209.[Abstract/Free Full Text]

Ventura, M., Canchaya, C., van Sinderen, D., Fitzgerald, G. F. & Zink, R. (2004c). Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon, its genetic structure, characterization and phylogenic analysis. Appl Environ Microbiol 70, 3110–3121.[Abstract/Free Full Text]

Ventura, M., Zink, R., Fitzgerald, G. F. & van Sinderen, D. (2005a). Gene structure and transcriptional organization of the dnaK operon of Bifidobacterium breve UCC 2003 and its application in bifidobacterial tracing. Appl Environ Microbiol 71, 487–500.[Abstract/Free Full Text]

Ventura, M., Fitzgerald, G. F. & van Sinderen, D. (2005b). Genetic and transcriptional organization of the clpC locus in Bifidobacterium breve UCC 2003. Appl Environ Microbiol (in press).

Vriend, G. (1990). WHAT IF: a molecular modeling and drug design program. J Mol Graph 8, 52–56.[CrossRef][Medline]

Wawrzynow, A., Baneckim, B. & Zylic, M. (1996). The Clp ATPases define a novel class of molecular chaperones. Mol Microbiol 21, 859–899.[CrossRef]

Wickner, S., Maurizi, M. R. & Gottesman, S. (1999). Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888–1893.[Abstract/Free Full Text]

Wolf, A., Kramer, R. & Morbach, S. (2003). Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC 13032 and their significance in response to osmotic stress. Mol Microbiol 49, 1119–1134.[CrossRef][Medline]

Yuan, G. & Wong, S. L. (1995). Regulation of groE expression in Bacillus subtilis: the involvement of the {sigma}A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol 177, 5427–5433.[Abstract/Free Full Text]

Received 6 May 2005; revised 13 June 2005; accepted 17 June 2005.



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