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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY837845, AY837846 and AY842855.
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INTRODUCTION |
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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 (2122 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 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.
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METHODS |
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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 [
-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 [-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 cm1 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 profileprofile 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.
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RESULTS |
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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 180413) and ATP-binding 2 (amino acids 545719), 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 414544), 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. 2
a), 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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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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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-value1010) 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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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 proteinDNA 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. 6
a). 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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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).
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DISCUSSION |
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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 (
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 (
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.
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ACKNOWLEDGEMENTS |
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Received 6 May 2005;
revised 13 June 2005;
accepted 17 June 2005.
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