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Journal of Bacteriology, August 2005, p. 5799-5808, Vol. 187, No. 16
0021-9193/05/$08.00+0     doi:10.1128/JB.187.16.5799-5808.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Proteomic Analysis of Global Changes in Protein Expression during Bile Salt Exposure of Bifidobacterium longum NCIMB 8809

Borja Sánchez,1,2 Marie-Christine Champomier-Vergès,1* Patricia Anglade,1 Fabienne Baraige,1 Clara G. de los Reyes-Gavilán,2 Abelardo Margolles,2 and Monique Zagorec1

Unité Flore Lactique et Environnement Carné, INRA, Domaine de Vilvert, 78350 Jouy-en-Josas, France,1 Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (CSIC), Ctra. Infiesto s/n, 33300, Villaviciosa, Asturias, Spain2

Received 26 January 2005/ Accepted 19 May 2005


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ABSTRACT
 
Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g liter–1) and a major (1.2 g liter–1) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.


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INTRODUCTION
 
Bifidobacteria are natural members of the human intestinal microbiota, in which they occur at concentrations of 109 to 1011 cells per g of feces, and represent up to 91% of the total gut population during the early stages of life (20). Indigenous bacteria of the gastrointestinal tract have evolved to tolerate stress factors. In this way, enteric bacteria are able to respond to the bactericidal effects of bile salts, which are detergent-like biological substances that disrupt the lipid bilayer structure of cellular membranes, induce protein misfolding, and cause oxidative damage to DNA (3). The mechanisms allowing intestinal bacteria to resist physiological bile concentrations remain poorly understood and have been mainly related to bile salt-modifying enzymes (27) or to membrane proteins that either take up or extrude these compounds (10, 54, 64). These adaptive mechanisms are very important to ensure the adaptation of bifidobacteria to the intestinal environment.

Some Bifidobacterium strains are being included as probiotic active ingredients in functional foods, mainly dairy products. Some health-promoting effects, like treatment of diarrhea and balancing of the intestinal microbiota, have been clinically established for some strains of this genus (45), and many others, such as antimutagenic and anticarcinogenic activity increase of the immune response and reduction of serum cholesterol levels, have been proposed (24).

Bifidobacterium longum is one of the bacterial species which are commonly isolated from adult and infant feces (18). It is particularly well adapted to the colonic environment, as reflected by its broad range of utilization of oligosaccharides (47, 48) and its ability to adapt to high concentrations of bile salts (39), which are present in the gut at concentrations usually below 5 mM (50).

It has been shown that some gram-positive bacteria can develop an adaptive response when subjected to moderate stress conditions. This response usually involves multiple genes, as deduced from quantitative changes detected in the mRNA and protein contents throughout cell life (6, 8, 55). Synthesis of molecular chaperones, which promotes proper protein folding, is a common response to some stress stimuli. Genes coding for these proteins, such as groES, groEL, and dnaK, have been shown to be inducible by osmotic and heat shock in Bifidobacterium breve (57, 58). Furthermore, sublethal bile concentrations can also trigger a physiological adaptive response in bifidobacteria (7, 23, 28), being DnaK induced in some species of Bifidobacterium in the presence of bile (49). Marvin-Guy et al. (36) recently used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for rapid identification of bile salt stress-related fingerprints from whole Bifidobacterium lactis cells, but no information was provided about protein identification and function. Furthermore, a proteomic study of Bifidobacterium infantis generated by multidimensional chromatography coupled to tandem mass spectrometry led to the identification of 136 proteins of this species (59). However, an exhaustive analysis of the molecular mechanisms of the cellular response to bile stress had not been performed yet.

Recently, the genome of B. longum NCC2705 has been sequenced (48). In order to efficiently exploit this information, we used a proteomic approach to analyze the regulation of bifidobacterial proteins during growth in the presence of bile salts by separation through two-dimensional (2D) electrophoresis and subsequent identification by MALDI-TOF mass spectrometry. B. longum NCIMB 8809, a human isolate with the capacity to produce an antimicrobial substance (40), was chosen as a model microorganism for this study. To the best of our knowledge, this is the first report dealing with the Bifidobacterium response to bile at a molecular level and constitutes the first proteomic analysis of this genus under stress conditions.


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MATERIALS AND METHODS
 
Bacterial strain and growth conditions. The strain used in this study was B. longum NCIMB 8809 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, United Kingdom), which was originally isolated from nursling stools. When required, it was subcultured on MRS agar plates (BD Diagnostic Systems, Sparks, MD) supplemented with 0.05% (wt/vol) L-cysteine (MRSC; Merck KGaA, Darmstadt, Germany). Bacteria were incubated at 37°C in an anaerobic chamber (Bactron Anaerobic/Environmental Chamber, Sheldon Manufacturing Inc., Cornelius, OR) in an atmosphere of 5% CO2-5% H2-90% N2. Liquid cultures were performed in 250 ml MRSC supplemented with two different bile salt concentrations (ox bile extract LS55 [Oxoid Limited, Hampshire, United Kingdom] at 0.6 and 1.2 g liter–1 [1.2 and 2.4 mM, respectively]). The supplemented MRSC was inoculated at 1% (vol/vol) with overnight cultures and then incubated anaerobically and monitored spectrophotometrically at 600 nm. Three independent cultures were made for each condition. Growth rates (µ) were estimated from the growth curve by fitting the data to the equation Nt = N0 x eµ xt, in which Nt and N0 are the cell densities at time t and time zero, respectively.

Extraction of cell-free proteins. Cultures of 250 ml grown to mid-exponential phase (optical density at 600 nm [OD600], 0.6; approximately 7 x 108 CFU/ml) were centrifuged at 2,800 x g at 4°C and washed twice in 0.1 M Tris-HCl buffer, pH 7.5, for 15 min. Bacterial pellets were resuspended in 5 ml of 1 M Tris-HCl buffer, pH 7.5, and broken by a single pass through a cell disrupter (Basic Z; Constant Systems Ltd., Daventry, United Kingdom) at 2.5 x 105 Pa. Unbroken cells and cell debris were removed by centrifugation at 4,500 x g for 15 min at 4°C. Membrane vesicles were discarded from the solution by ultracentrifugation at 50,000 x g for 30 min at 4°C. The protein concentration was estimated using the Bradford method according to the manufacturer's instructions (Coomassie Protein Assay Reagent; Pierce Biotechnology, Rockford, IL).

2D electrophoresis conditions. Bifidobacterial proteins were analyzed by methods originally derived from previously described methods (1, 35). Proteins (350 µg) were treated for 30 min at 37°C by 1 µl Benzonase (Merck KGaA) in the presence of 10 mM MgSO4 to remove nucleic acids. Proteins were then precipitated by methanol-chloroform (3:1, vol/vol). The mixture was vortexed and centrifuged at 2,800 x g for 2 min at room temperature. Four volumes of deionized water was added, and after vigorous vortexing and further centrifugation at 2,800 x g for 2 min at room temperature, the upper phase was removed and proteins were precipitated by the addition of 3 volumes of methanol and centrifugation at 2,800 x g for 4 min. The pellet of proteins was dried at 95°C and solubilized in 40 µl of a mixture containing 6.5 M deionized urea, 2.17 M thiourea (Amersham Biosciences, Buckinghamshire, United Kingdom), 65 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich, St. Louis, MO), 100 mM dithiothreitol, and 25 mM Tris-HCl buffer, pH 8.8 (Amersham Biosciences). The volume was adjusted to 380 µl with a solution containing 6.5 M deionized urea, 2.17 M thiourea, 65 mM CHAPS, 100 mM dithiothreitol, 0.5% pH 4 to 7 carrier ampholytes (Bio-Rad Laboratories, Hercules, CA), and 2 µg of bromophenol blue. This solution was used to rehydrate 17-cm pH 4 to 7 linear immobilized pharmalyte gradient strips (Bio-Rad). Strips were rehydrated for 12 h at 50 V using the Protean IsoElectric Focusing Cell II (Bio-Rad) and then focused at 60,000 V/h. Focused immobilized pharmalyte gradient strips were equilibrated sequentially for 15 min in a buffer (1 M Tris-HCl, pH 6.8, containing 6 M urea, 30% [vol/vol] glycerol [Merck KGaA]) and 1% (wt/vol) sodium dodecyl sulfate [Merck KGaA]) supplemented with 0.83% (wt/vol) dithiothreitol in the first equilibration step and with 7.5% (wt/vol) iodoacetamide in the second one. The second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on gels containing 12.5% polyacrylamide and carried out with a Protean II xi cell (Bio-Rad), and proteins were resolved at a constant current of 10 mA/gel overnight at 4°C. Proteins were visualized following staining with Bio-Safe Coomassie (Bio-Rad) according to the manufacturer's instructions. Spot detection and volume quantitation were carried out with ImageMaster 2D Elite (version 3.10; Amersham Biosciences). At least three independent experiments for each growth condition were performed. An effect of bile salts on the expression of proteins was considered if the mean normalized spot volume varied at least 1.9-fold and confirmed by analysis of variance at a significance level of P < 0.05 using the bile salt concentration as a factor with three categories: 0, 0.6, and 1.2 g liter–1. This quantification of the variation in spot intensity was named the induction factor.

Identification of proteins by peptide mass fingerprinting. Individual spots were excised from gels and submitted to tryptic digestion, and mass spectrometry analyses were performed as previously described (19). MS-Fit (University of California San Francisco Mass Spectrometry Facility; http://prospector.ucsf.edu) and Mascot (Matrix Science Inc., Boston, MA; http://www.matrixscience.com/search_form_select.html), installed locally, were used to identify proteins from peptide mass fingerprints. All searches were performed against the database for B. longum NCC2705 (http://www.ncbi.nlm.nih.gov/genomes/).

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (F6PPK) assay, glucose consumption, and organic acids production. Cell extracts were obtained from Bifidobacterium cultures (10 ml) harvested at an OD600 of 0.6 by centrifugation (10,000 x g for 15 min) and washed with 10 ml of 100 mM potassium phosphate buffer, pH 7.0. The pellet was resuspended in 1 ml of the same buffer. Cells were sonicated while cooling on ice using a CV17 sonicator (VibraCell, Newtown, CT) and centrifuged to remove cell debris. F6PPK activity was measured spectrophotometrically as the ferric acetyl hydroxamate produced from the enzymatically generated acetyl phosphate, according to Sánchez et al. (46). One unit of activity was defined as the amount of protein that releases 1 nmol of acetyl phosphate per min. Specific activity was expressed as units mg protein–1.

For glucose consumption and organic acid determination, studies were performed with buffered cell suspensions, which greatly facilitated product analysis. Ten milliliters of Bifidobacterium cultures at an OD600 of 0.6, grown in the absence or presence (0.6 or 1.2 g liter–1) of bile salts, were collected by centrifugation (10,000 x g for 15 min). The pellet was washed twice with 10 ml 33 mM potassium phosphate buffer, pH 7.0, and resuspended in 10 ml of 33 mM potassium phosphate buffer, pH 5.6, containing 25 mM glucose. The suspension was incubated with constant mild stirring for 4 h at 37°C. Cells were removed from the suspension by centrifugation, and the supernatant was analyzed by high-performance liquid chromatography to quantify glucose and organic acid levels. An Alliance 2690 automatic injection system (Waters Corporation, Milford, MA) was used. Samples (50 µl) were isocratically separated at a flow rate of 0.7 ml/min at 65°C on an HPX-87H Aminex ion-exchange column (300 by 7.8 mm; Hewlett-Packard, Palo Alto, CA) protected by a cation H+ Microguard cartridge (Bio-Rad) using 3 mM sulfuric acid as the mobile phase. A photodiode array PDA 996 detector (Waters) was used for quantification of organic acids at 210 nm, whereas glucose was quantified with a 410 differential refractometer detector. For the three growth conditions assayed (absence of bile and presence of 0.6 and 1.2 g liter–1 of bile), the acetic to lactic acid molar ratios were calculated. Results presented are the means of at least three separate experiments.

Data on F6PPK activity levels, glucose consumption, and organic acid formation by resting cells were subjected to one-way analysis of variance with the SPSS 11.0 software for Windows (SPSS Inc., Chicago IL) using the factor "concentration of bile salts" with three categories: 0, 0.6, and 1.2 g liter–1. The least-significant-difference test was used for comparison of means.


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RESULTS AND DISCUSSION
 
Growth of B. longum NCIMB 8809 in the presence of bile salts. The ability to grow in the presence of bile salts was tested. B. longum NCIMB 8809 was grown in batch cultures to determine the times it takes to reach mid-log phase and stationary phase (Fig. 1). While growth rates were very similar during the exponential phase (0.57, 0.53, and 0.50 at bile salt concentrations of 0, 0.6, and 1.2 g liter–1, respectively), the logarithm of CFU/ml reached during stationary phase differed among the three cultures, being 9.88 ± 0.07 in the absence of bile and 9.44 ± 0.05 and 9.18 ± 0.04 in the presence of 0.6 and 1.2 g liter–1, respectively. Therefore, 0.6 and 1.2 g liter–1 bile salts were considered sublethal concentrations in the present study.



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  		FIG. 1. Growth of B. longum NCIMB 8809 in MRSC broth with 0 (•), 0.6 ({circ}), and 1.2 ({blacktriangledown}) g liter–1 added bile salts. Error bars represent the standard deviations for triplicate experiments.

A proteomic approach allowed the detection of differences in protein expression patterns of B. longum grown in the presence of different concentrations of bile salts. Proteins that could play a role in the ability of B. longum to grow in the presence of bile salts were identified in this study. The genome data of the 2.26-Mb chromosome of B. longum NCC2705 (48) were used to assign putative genes encoding the proteins experimentally obtained from B. longum NCIMB 8809 extracts by comparison of peptide mass fingerprinting to the database. Our study focused on the cytosolic proteins in a pI range of 4 to 7 expressed under bile salt stress conditions. Protein expression was determined using cells grown in the absence of bile salts as the standard condition and compared with the patterns of extracts of cells grown in the presence of 0.6 or 1.2 g liter–1 bile salts. In this study, we focused only on spots which were considered to be up- or down-regulated by a factor of greater than 1.9 in the presence of bile salts. Figure 2 shows 2D gels of cell protein extracts of B. longum grown either in the absence or in the presence of 0.6 or 1.2 g liter–1 bile salts. Comparison of protein patterns obtained under the three conditions revealed that 34 spots clearly displayed different levels of expression, depending on the bile salt concentrations present in the growth medium. Among these proteins, 26 were overexpressed, including 14 which were significantly overexpressed at 0.6 g liter–1 bile salts and 22 which were significantly overexpressed at 1.2 g liter–1 bile salts. Only five spots were less expressed in the presence of bile salts, four of them being significantly down-regulated at bile salt concentrations of both 0.6 and 1.2 g liter–1. Finally, three spots were detected only when bile salts were added to the medium and were absent under the other conditions.



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  		FIG. 2. 2D gels showing the proteins expressed in MRSC medium without bile salts (a), with 0.6 g liter–1 bile salts (b), or with 1.2 g liter–1 bile salts (c). MM, molecular mass.

These 34 spots were excised from gels and subjected to peptide mass fingerprinting analysis in order to identify their putative genes and to deduce their putative functions (Table 1).


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  		TABLE 1. Identification of B. longum NCIMB 8809 proteins affected by bile salt exposure

Ox bile extract promotes induction of general stress response proteins. Genes involved in the general stress response are highly conserved among eubacteria. They include a large set of proteins which are generally involved in the maturation of newly synthesized proteins, in protein targeting to membranes, in refolding and degradation of denatured proteins, and in DNA repair (14, 16). Two such chaperone proteins frequently involved in these processes (spots BL15 and BL64, identified as GroEL and DnaK, respectively; Table 1) were found to be overexpressed in B. longum in the presence of bile salts. Their levels of induction were higher at 0.6 g liter–1 than at 1.2 g liter–1, which suggests that low basal levels of bile salts could be enough to trigger the response mediated by these two chaperones. DnaK and GroEL have been frequently related to the response against several environmental stimuli. Induction of dnaK at the transcriptional level was shown in Bifidobacterium adolescentis following heat, salt, and bile treatments (49). DnaK was induced in Propionibacterium freudenreichii, a microorganism closely related to bifidobacteria, as a response to bile, acid, and heat (32, 33) and in B. breve as a response to osmotic shock (58). GroEL clearly responds to acid stress in P. freudenreichii (33) and Streptococcus oralis (61). It has also been shown that GroEL is induced during heat adaptation of different gram-positive microorganisms such as Bacillus subtilis (43), Staphylococcus aureus (30), and B. breve (57). DnaK and GroEL are induced by bile in Enterococcus faecalis (12, 15), by salt stress in Lactococcus lactis (26), by heat stress in Bacillus cereus and Lactobacillus delbrueckii subsp. bulgaricus (17, 42), and by acid stress in L. lactis (21).

On the other hand, spot BL68, only detected when the bile salt concentration was 1.2 g liter–1, was identified as a response regulator of a two-component signal transduction system (Table 1). These systems are involved in the stress response to bile salts in P. freudenreichii (32). In the genome of B. longum NCC2705, the gene encoding this protein is followed by the corresponding histidine kinase gene and by a gene for a hypothetical protein similar to the ImpA protein of Salmonella typhimurium. ImpA is involved in the protection of DNA against UV damage (34) and shows homology with SOS response transcriptional regulators. It was shown in Escherichia coli that promoters most consistently induced by bile salts were linked to genes whose products help the cell to cope with oxidative stress or to respond to DNA damage (3). In Lactobacillus plantarum, five bile-inducible genes are annotated as having functions involved in redox reactions (4). In addition, mammalian cells, when exposed to bile salts, also activate stress response proteins that respond to oxidative stress and DNA damage (41), suggesting that these two deleterious effects may be general consequences of the action of bile on cells.

Several enzymes involved in carbohydrate catabolism, including the key enzyme of the so-called bifidobacterial shunt, are overexpressed in cells grown in the presence of bile salts. Thirteen proteins related to carbohydrate metabolism were induced by bile salt exposure, and one of them was detected only when cells were grown at the highest bile extract concentration we tested, 1.2 g liter–1 (Table 1). Among the glycolytic enzymes, we observed the overexpression of BL81, identified as Xfp (F6PPK), the key enzyme of the so-called bifidobacterial shunt, a unique hexose metabolism that occurs via the phosphoketolase pathway in the genus Bifidobacterium (37). This dual enzyme catalyzes the conversion of fructose 6-phosphate to erythrose 4-phosphate and also acts on xylulose 5-phosphate, producing acetyl-phosphate and glyceraldehyde-3-phosphate (Fig. 3). In a previous study, we reported that the acquisition of stable resistance to bile salts in bifidobacteria increases F6PPK activity (46). In the present work, we found that Xfp was overexpressed with an induction factor of 3.71 when B. longum was grown in 1.2 g liter–1 bile salts, most likely indicating that this enzyme plays a crucial role not only in bile salt adaptation but also in protection of the cell against bile salt stress.



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  		FIG.3. Schematic representation of the carbon catabolic pathway and the bifidobacterial shunt in B. longum. Influence of bile on the expression of the enzymes catalyzing the different steps is indicated by white (no bile present), gray (0.6 g liter–1 bile salts), and black (1.2 g liter–1 bile salts) bars. Normalized volume ratios greater than 4 are shown with a height break. Most of the enzymes were up-regulated by bile salt addition. Pfl was only detected when a 1.2 g liter–1 bile salt concentration was present in the medium (*). Eno was not detected under any conditions (ND), and Tal was not up-regulated (NDE). Abbreviations: AckA, acetate kinase; Adh2, aldehyde-alcohol dehydrogenase 2; Eno, enolase; Gap, glyceraldehyde-3-phosphate dehydrogenase C; GlkA, glucokinase; Gpi, glucose 6-phosphate isomerase; Gpm, phosphoglycerate mutase; Ldh2, lactate dehydrogenase; Pgk, phosphoglycerate kinase; Pfl, formate acetyltransferase; Pyk, pyruvate kinase; Tal, transaldolase; Tkt, transketolase; Xfp, F6PPK; CoA, coenzyme A; P, phosphate.

BL26 was identified as glyceraldehyde-3-phosphate dehydrogenase C (Gap), the enzyme that catalyzes the interconversion of glyceraldehyde-3-phosphate and glyceraldehyde-1,3-bisphosphate. Gap is a major controlling point of carbon flux in lactic acid bacteria (63) and has been shown to be rate limiting in the glycolytic activity of starved L. lactis cells (44). Interestingly, its induction is dependent on the bile salt concentration in B. longum NCIMB 8809 and the level of its induction was the highest among the proteins analyzed in this work (9.29 at 0.6 g liter–1 bile salts and 13.95 at 1.2 g liter–1 bile salts).

Several spots corresponding to additional proteins involved in the transformation of glyceraldehyde-3-phosphate to pyruvate (BL21, BL34, and BL16, identified, respectively, as the phosphoglycerate kinase Pgk, the phosphoglycerate mutase Gpm, and the pyruvate kinase Pyk) were also detected at higher levels when cells were grown in the presence of bile salts. These proteins have also been reported to be induced under environmental stress conditions in other microorganisms (9, 13, 31, 65). Remarkably, for enolase, the enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate, no significant variation was observed under bile salt exposure at the conditions assayed in this work although our previous studies suggested that it could be overexpressed in a bile-adapted Bifidobacterium strain (46).

Furthermore, four spots corresponding to proteins potentially involved in pyruvate catabolism were induced when bile salts were present in the growth medium. BL31 and BL40 were identified as lactate dehydrogenase (Ldh2) and acetate kinase (AckA), respectively. These two enzymes are involved in the formation of lactate and acetate, the two major metabolic end products of the bifidobacterial shunt. Lactate dehydrogenase has been shown to be affected by various stresses in E. faecalis (15). In addition, BL2 was identified as aldehyde-alcohol dehydrogenase 2 (Adh2), which, in bifidobacteria, could be involved in the interconversion of both ethanol and acetaldehyde and of acetaldehyde and acetyl coenzyme A, with NADH production in both reactions. The fourth spot, BL66, was identified as formate acetyltransferase (Pfl), the enzyme that catalyzes the conversion of formate to pyruvate in the presence of acetyl coenzyme A. We found this enzyme to be a specific bile salt response protein in B. longum NCIMB 8809 since it was detected only in the presence of 1.2 g liter–1 bile salts. Pfl has also been reported to be induced by acid stress in S. oralis (61).

The synthesis of several glycolytic enzymes increased markedly in the presence of bile salts, suggesting a global activation of the glycolytic pathway with enhanced production of energy-rich intermediates and reducing equivalents. Such an activation of some glycolytic enzymes as a response to a low pH has also been reported previously in S. oralis by Wilkins et al. (61). The possibility that a broad activation of the carbohydrate catabolism of B. longum could have been produced under bile stress is supported by the induction of additional enzymes. Indeed, we observed induction of glycogen phosphorylase (BL4, GlpP), which hydrolyzes (1,4-{alpha}-D-glucosyl)n compounds in the presence of Pi, and of UDP-glucose 4-epimerase (BL29, GalE1), which catalyzes the interconversion of UDP-glucose and UDP-galactose and which could be involved in the utilization of carbon sources other than glucose during growth. In this respect, it is noteworthy that nutrient limitation and heat shock caused the activation of glycogen metabolism in yeast (52). Moreover, we identified spot BL7 as transketolase (Tkt). This enzyme is responsible for the formation of xylulose 5-phosphate and can fuel the glycolytic pathway through the bifidobacterial shunt. BL20 was identified as the 6-phosphogluconate dehydrogenase decarboxylatin II (Gnt), an enzyme transforming 6-phospho-D-gluconate to D-ribulose 5-phosphate in the pentose phosphate pathway, with the formation of NADPH. Gnt has been reported to be induced by salt stress in rice shoots (22). Finally, it is worth noting the overexpression of BL24 (MliK), annotated in the genome of B. longum NCC2705 as the cytoplasmic part of an ABC transporter for sugars, which points to the activation of sugar transport as a response to bile challenge. A schematic representation of the influence of bile on the enzyme levels of glycolysis and pyruvate metabolism is depicted in Fig. 3.

Ox bile extract promotes changes in carbohydrate catabolism. As stated above, and based on 2D electrophoresis results, several proteins related to sugar metabolism were found to be overexpressed after bile extract exposure. Therefore, to support the proteomic approach with physiological data, we analyzed the influence of different bile salt concentrations on the production of end metabolic products (lactate, acetate, and formate) and F6PPK activity. This activity on fructose 6-phosphate is one of the most common taxonomic tests for this genus, since it is usually not present in other gram-positive intestinal bacteria and is mediated by Xfp, the first enzyme of the phosphoketolase pathway, which was found to be upregulated in the presence of bile salts (Table 1). As we expected, F6PPK activity was significantly higher in protein extracts of cells grown in the presence of bile (Table 2), although the activity did not exactly correlate with the Xfp protein levels. This can be due to the dual nonspecific activity of Xfp, acting also on xylulose 5-phosphate (37). In addition, several enzymes of pyruvate catabolism were also found to be overexpressed (Table 1). High-performance liquid chromatography analyses corroborated that when resting cells were incubated in a glucose-containing buffer, they consumed significantly more sugar and produced more lactic and acetic acids if bile salts were present in the growth medium (Table 2). Moreover, the acetic/lactic acid ratio decreased in the presence of bile, strongly supporting the hypothesis of global activation of the glycolytic pathway, which subsequently enhanced the formation of lactic acid. In contrast, no formic acid was detected, as should be expected from the expression of the enzyme Pfl in the presence of bile. These results could be explained by the short incubation time of resting cells in the glucose-containing buffer (4 h) and the early stage of growth of the cultures analyzed. In this respect, van der Meulen et al. (56) found that young cultures of Bifidobacterium animalis do not produce formic acid, which is indeed formed in older cultures at the expense of lactic acid production. In our case, when bile is present, an accumulation of intermediate compounds of the glycolytic pathway could occur in metabolically active cultures at early stages of growth, which could lead to the formation of larger amounts of end products, including formic acid, when cultures were older. These findings strongly suggest that bile salts have an impact on the carbon metabolic fluxes in B. longum.


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  		TABLE 2. F6PPK specific activities of cell-free extracts, glucose consumed, formation of acetic and lactic acids, and acetic/lactic acid ratios from cell suspensions of B. longum NCIMB 8809 grown in the absence or presence of different bile salt concentrationsa

Regulation by bile salts of proteins involved in transcription, translation, and metabolism of amino acids and nucleotides. Proteins involved in transcription and translation were also found to have a regulated expression following bile salt exposure (Table 1). Several tRNA synthetases, or any of their components (BL8, BL17, BL19, and BL42), were overexpressed in the presence of bile salts. BL50 was identified as a ribosomal recycling factor acting on disassembly of the termination complex and prevention of translation errors (60). Its repression was dependent on the bile salt concentration added to the growth medium; the lowest production was observed at the highest bile salt concentration (1.2 g liter–1). BL82 (slightly overexpressed) and BL51 (repressed) were identified as elongation factors Tu and P, respectively. These proteins are involved in the delivery of aminoacyl-tRNA to the ribosome (51). Elongation factor Tu has been described as a chaperone on unfolded and denatured proteins in E. coli (5) and has been reported to be induced during acid adaptation in P. freudenreichii and S. mutans (33, 62).

Two ribosomal proteins were identified. BL80, the 30S ribosomal protein S2, was induced, whereas BL38, probably 50S ribosomal protein L25, was repressed. Some 30S ribosomal proteins have been previously detected after salt stress in Listeria monocytogenes (9) and after phosphate and amino acid starvation, as well as after ethanol, heat, and salt stresses, in B. subtilis (2, 11). BL27, identified as the {alpha} subunit of the DNA-directed RNA polymerase which is required for the assembly of the enzyme and the interaction with some regulatory proteins (25), appeared to be overexpressed after bile salt exposure. BL56 was identified as the N utilization substance homolog NusA. This protein is crucial in ensuring that coupled transcription-translation takes place in E. coli and is necessary for optimal activity of the N-dependent antitermination system (29, 53). Surprisingly, it was repressed at 0.6 g liter–1 bile salts and overexpressed at a higher bile salt concentration (1.2 g liter–1) compared to the standard conditions.

Relating to amino acid metabolism, spot BL36 was identified as the methionine aminopeptidase that removes the N-terminal methionine from nascent proteins and was detected in smaller amounts when bile salts were added to the culture medium. A NifS-like aminotransferase (BL41) was detected only when B. longum was grown in the presence of bile salts. All these proteins are thought to be involved in the assembly of iron-sulfur clusters (66), and it has been shown that NifS-like protein is induced in Mycobacterium smegmatis when the cells enter the anaerobic stationary phase (38). It is therefore possible that some iron-sulfur proteins could play a role in the growth of B. longum in the presence of bile, perhaps modifying the anaerobic environment. On the other hand, BL44 was identified as a probable branched-chain amino acid aminotransferase (IlvE), which catalyzes the last step of the synthesis of the hydrophobic amino acids valine, leucine, and isoleucine. The induction of ilvE points to an increase in the synthesis of hydrophobic amino acids that could be needed for building proteins with high hydrophobicity.

Finally, three enzymes involved in nucleotide biosynthetic pathways were also identified. BL9 and BL11 were detected in larger amounts when bile salts were present in the medium and were identified as the enzymes CTP synthase and PurH, which are involved in cytosine and purine synthesis, respectively. The protein BL39 was identified as adenylate kinase, an enzyme involved in adenine synthesis. This reversible enzyme provides a way to obtain ATP from ADP, as well as to convert AMP to ADP. Its repression by bile salts, together with the activation of the glycolytic pathway, supports the hypothesis that ATP synthesis could be redirected mainly to substrate level phosphorylation reactions through the bifidobacterial shunt as a response to bile stress.

Conclusion. The present work constitutes a first insight into the molecular mechanism leading to efficient bile tolerance in intestinal bifidobacteria and establishes the first proteomic profiles for these bacteria under intestinal stress conditions. Thirty-four protein spots of B. longum NCIMB 8809 detected on 2D gels could be identified by the use of the genome sequence of B. longum NCC2705. The intensity of these spots was increased or decreased, depending on the presence of bile salts, mimicking the bile stress encountered by bifidobacteria in the human gastrointestinal tract. Our data clearly demonstrate that the expression of proteins from different functional categories is modulated as a result of the exposure of B. longum to bile salts. Some of these proteins involve general stress response proteins, but others are key components of the central and intermediary metabolism, including the transcription-translation machinery, gene regulation, and protein synthesis. Remarkably, the activation of glycolysis and pyruvate catabolism suggests a shift of the bacterial metabolism to render more reducing equivalents and energy-rich intermediates, such as ATP. This activation would be probably focused on the enhancement of cellular processes that protect bifidobacteria against the deleterious action of bile salts such as oxidative stress, DNA damage, and intracellular pH acidification. The present findings suggest that, rather than a single event, the response to bile challenge involves a complex physiological mechanism. Recent results from our research group indicate an increase in membrane-bound H+-ATPase activity in a strain of Bifidobacterium in response to bile (data not shown), suggesting that the extra ATP coming from the increased glycolytic flux could be used to feed this enzyme and therefore maintain the internal pH of the cell under the stress conditions promoted by bile. This proteomic analysis-based approach has revealed a number of targets and molecular markers for future genetic and physiological studies and has certainly shed new light on the mechanism involved in the bile adaptation and tolerance of these intestinal bacteria.


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ACKNOWLEDGMENTS
 
This work was financed by European Union FEDER funds and the Spanish Plan Nacional de I + D (projects AGL2001-2296 and AGL2004-06727-C02). B. Sánchez was the recipient of a predoctoral fellowship from the Spanish Ministerio de Ciencia y Tecnología and from the MICA Department of the National Institute for Research in Agronomy (INRA). MALDI-TOF experiments were performed on the PAPPS platform of the INRA Center at Jouy-en-Josas.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unité Flore Lactique et Environnement Carné, INRA, Domaine de Vilvert, 78350 Jouy-en-Josas, France. Phone: 33 (0)1 34 65 22 92. Fax: 33 (0)1 34 65 21 05. E-mail: Marie-Christine.Champomier-Verges{at}jouy.inra.fr. Back


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Journal of Bacteriology, August 2005, p. 5799-5808, Vol. 187, No. 16
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