Genetic Characterization of the Bile Salt Response in Lactobacillus plantarum and Analysis of Responsive Promoters In Vitro and In Situ in the Gastrointestinal Tract

Abstract

In this paper we describe the growth, morphological, and genetic responses of Lactobacillus plantarum WCFS1 to bile. Growth experiments revealed that a stepwise increase in the porcine bile concentration led to a gradual decrease in the maximal growth rate. Moreover, the final density reached by an L. plantarum culture growing in MRS containing 0.1% bile was approximately threefold lower than that in MRS lacking bile. The morphology of the cells grown in MRS containing 0.1% bile was investigated by scanning electron microscopy, which revealed that cells clumped together and had rough surfaces and that some of the cells had a shrunken and empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of L. plantarum cells grown in MRS without bile. An alr complementation-based genome-wide promoter screening analysis was performed with L. plantarum, which led to identification of 31 genes whose expression was potentially induced by 0.1% porcine bile. Remarkably, 11 membrane- and cell wall-associated functions appeared to be induced by bile, as were five functions involved in redox reactions and five regulatory factors. Moreover, the lp_0237 and lp_0775 genes, identified here as genes that are inducible by bile in vitro, were previously identified in our laboratory as important for L. plantarum in vivo during passage in the mouse gastrointestinal tract (P. A. Bron, C. Grangette, A. Mercenier, W. M. de Vos, and M. Kleerebezem, J. Bacteriol. 186:5721-5729, 2004). A quantitative reverse transcription-PCR approach focusing on these two genes confirmed that the expression level of lp_0237 and lp_0775 was significantly higher in cells grown in the presence of bile and cells isolated from the mouse duodenum than in cells grown on laboratory medium without bile.

After ingestion bacteria meet several biological barriers, the first of which is the gastric acidity encountered in the stomach of the host. Bacteria able to survive these harsh conditions transit to the intestine, where they encounter stresses associated with low oxygen availability, bile salts, and competition with the microbiota. Bile salts are synthesized in the liver by conjugation of a heterocyclic steroid derived from cholesterol (17). The resulting conjugated bile salts are stored and concentrated in the gall bladder during the fasting state, and after consumption of a fat-containing meal these compounds are released into the duodenum, where they play a major role in the dispersion and absorption of fats, including bacterial phospholipids and cell membranes (34). Bile salts are reintroduced in the liver following their reabsorption in the distal small intestine and colon after deconjugation by the microbiota (16). This deconjugation reaction is performed by bacterial bile salt hydrolases, which are encoded in the genomes of several intestinal bacteria, including Bifidobacterium and Lactobacillus species (7, 10, 19, 33).

Studies of gram-positive, food-associated bacteria and their tolerance to digestive stress have focused mainly on physiological aspects, such as determination of the levels of acid and bile salt tolerance (6, 18), as well as the development of complex media in order to selectively enrich the bacteria that are digestive stress tolerant (30). Additionally, in several studies workers have described defense mechanisms of gram-negative bacteria against bile acids, which include the synthesis of porins, transport proteins, efflux pumps, and lipopolysaccharides (15). A few genome-wide approaches with gram-positive bacteria aimed at identification of proteins important for bile salt resistance have been described. In Propionibacterium freudenreichii, Listeria monocytogenes, and Enterococcus faecalis two-dimensional gel electrophoresis led to identification of several proteins that were expressed more highly in the presence of bile salts than under control conditions (12, 20, 26). In P. freudenreichii these induced proteins were characterized further, which led to the identification of 11 proteins that are induced by bile stress. These proteins include general stress proteins, such as ClpB and the chaperones DnaK and Hsp20 (20). Analogously, a subset of the proteins identified in E. faecalis appeared to be inducible by multiple sublethal stresses, including heat, ethanol, and alkaline pH (27). The fact that general stress proteins are induced by bile is in agreement with the cross-protection against bile after thermal or detergent pretreatment that has been observed in several bacteria, including E. faecalis, L. monocytogenes, and Bifidobacterium adolescentis (2, 12, 29). Furthermore, random gene disruption strategies with L. monocytogenes and E. faecalis resulted in strains that were more susceptible to bile salts than the wild-type strains. Subsequent genetic analysis of the mutants revealed that the disrupted genes encode diverse functions, including an efflux pump homologue (2), and genes that may be involved in the biosynthesis of cell walls and fatty acids (3).

Lactic acid bacteria (LAB) are used extensively in the production of fermented food products. Because of the frequent consumption of dairy, vegetable, meat, and other fermented food products, large amounts of LAB are ingested. Moreover, LAB have the potential to serve as delivery vehicles for health-promoting or therapeutic compounds to the gastrointestinal tract (GI tract) (13, 31). One of the LAB, Lactobacillus plantarum, is encountered in many environmental niches, including dairy fermentations, meat fermentations, and a variety of vegetable fermentations (9, 11, 28). The complete 3.3-Mbp genome sequence of L. plantarum WCFS1 was recently determined (19). This strain is a single-colony isolate of strain NCIMB8826, which effectively survives passage through the stomach in an active form, reaches the ileum in high numbers compared to other strains, and is detectable in the colon (35). The availability of its genome sequence allows effective investigation of the genes and regulation mechanisms underlying the observed persistence of L. plantarum in the GI tract. In the research described here we focused on the genetic response of L. plantarum after exposure to a toxic concentration of bile acid molecules. Previously, utilization of alanine racemase as a promoter probe for the genome-wide identification of L. plantarum genes whose expression is induced by high salt conditions was described (5). Here the same system was utilized, which led to the identification of 31 genes induced by bile salts. Strikingly, two of the genes identified have previously been demonstrated to be induced in vivo in L. plantarum during passage through the mouse GI tract (4). In a quantitative reverse transcription-PCR analysis we focused on these two genes, demonstrating both their in vitro chromosomal induction by porcine bile and their in vivo expression in the duodenum in a mouse model.

MATERIALS AND METHODS

Bacterial strains, media, and growth conditions.

The bacterial strains, plasmids, and primers that were used in this study are listed in Table 1. L. plantarum WCFS1 (19) and its alr mutant derivative MD007 (23) were grown at 37°C in MRS (Difco, Molesey, Surrey, United Kingdom) without aeration. Various concentrations of d-cycloserine (from freshly prepared, filter-sterilized stock solutions), 5 μg of erythromycin per ml, and 200 μg of d-alanine per ml were added to MRS medium as indicated below.

TABLE 1.

Strains, plasmids, and primers used in this study and their relevant characteristics

Strain, plasmid, or primer	Relevant features	Reference
L. plantarum     strains
WCFS1	Wild type whose genome sequence is available	19
MD007	L. plantarum WCFS1 Δalr, d-alanine auxotroph	23
Plasmids
pNZ7120	Emr, pIL252 derivative containing Lactococcus lactis alr promoter probe	5
BI1 to BI96	PNZ7120 derivatives containing chromosomal L. plantarum fragments that were initially identified as inducible by 0.1% porcine bile	This study
Primers
PB1	5′-ACCGCTACGGATCACATC-3′	5
PB2	5′-CTCGGGGCAGTAAGACTA-3′	5
PB3	5′-GTGGTGAAGTTTTCATGG-3′	5
16S-fo2	5′-TGATCCTGGCTCAGGACGAA-3′	This study
16S-re2	5′-TGCAAGCACCAATCAATACCA-3′	This study
237-fo	5′-CTACTGATATGGTTGTCGGGAATTA-3′	This study
237-re	5′-ACGGGTGCGTAGAAGAAGC-3′	This study
775-fo	5′-GCTCTTGCACCGGATATCAA-3′	This study
775-re	5′-TTTCTTCTTCCCGTGACCAGT-3′	This study
1898-fo	5′-GTGGCGACGGTTCTTACCAT-3′	This study
1898-re	5′-CCCTGGAAGACCAATCGTGT-3′	This study
1027-fo	5′-CCATGATGGTGCTTCACAA-3′	This study
1027-re	5′-TCGTGGCAGCAGAGGTAATG-3′	This study

Scanning electron microscopy.

Nuclepore polycarbonate membranes (Costar, Cambridge, Mass.) with 1-μm pores were incubated for 30 min in a 0.01% poly-l-lysine solution in 0.1 M Tris-HCl buffer. Several drops of a logarithmically growing L. plantarum culture were positioned on the poly-l-lysine-coated membranes and incubated for 5 min at 100% humidity. After this the bacteria were fixed on the membranes for 30 min in 3% glutaraldehyde. Subsequently, the membranes were washed three times with Milli Q water (MQ), dehydrated with ethanol by using 30, 50, 70, and 90% ethanol and finally 100% ethanol three times, and critical point dried by the CO₂ method (Balzers CPD 020; Balzers Union, Liechtenstein). The dried membranes were mounted on sample holders by using carbon adhesive tabs (Electron Microscopy Sciences, Washington, D.C.). The sample holders were positioned inside a preparation chamber (CT 1500 HF; Oxford Instruments, Abingdon, Oxon, England). The samples were sputter coated with 10 nm of platinum and analyzed with a field emission scanning electron microscope (JEOL 6300F; JEOL, Tokyo, Japan) at 2.5 kV. Images were recorded digitally, and image processing was performed with Adobe PhotoShop 5.5.

Identification of bile salt-inducible loci by using the alr complementation library.

Construction and utilization of an L. plantarum promoter probe library for effective identification of conditionally active promoters were described previously (5). Here this library was exploited for identification of clones that contain bile salt-inducible L. plantarum chromosomal fragments. Appropriate dilutions of a −80°C stock of the library were immediately plated on MRS plates containing erythromycin and 0.1% porcine bile (B-8631; Sigma, Zwijndrecht, The Netherlands). After 2 days of growth approximately 4,000 individual colonies on these plates were used for replica plating on plates containing erythromycin, one with 0.1% porcine bile and one without porcine bile. Growth on the two plates was periodically compared, which led to the primary identification of 96 colonies that could grow only in the presence of porcine bile (Table 1). The colonies displaying bile-dependent growth were cultured overnight in a microtiter plate containing MRS with erythromycin and d-alanine. The full-grown cultures were used to reconfirm the initially observed conditional growth phenotype and to assess the conditional promoter strength by observing the conditional growth phenotype in the presence of different concentrations of the Alr inhibitor d-cycloserine (5). The microtiter plate was replica plated by using a 96-pin replicator and MRS plates containing erythromycin and 0, 2.5, 5.0, 10, 25, 50, 100, or 200 μg of d-cycloserine per ml, with or without 0.1% bile salts. The bile-dependent, differential growth phenotype in the presence of different concentrations of d-cycloserine was determined by periodically comparing the growth on the plates with bile salts and the growth on the plates without bile salts. For the clones for which bile-dependent growth could be confirmed in this experiment, the chromosomal L. plantarum inserts harbored by their pNZ7120 derivatives were amplified by PCR by using primers PB1 and PB2 (5). The resulting amplicons were used for partial DNA sequence analysis with primer PB3 (5), and the insert sequences determined were assigned to chromosomal loci by using BlastN (1) and the L. plantarum genome sequence as the database (19).

RNA isolation.

Appropriate dilutions of an overnight culture of L. plantarum WCFS1 were plated on MRS with or without 0.1% porcine bile salts. After 3 days of growth approximately 100 colonies were rapidly collected from the plates in 3 ml of MRS, which was added to 12 ml of quench buffer (60% methanol, 66.7 mM HEPES; pH 6.5; −40°C) (B. Pieterse, unpublished data). Following quenching, the cells were immediately pelleted by centrifugation at 5,000 × g for 10 min, and the cell pellets were resuspended in 0.4 ml of ice-cold MRS. The cell suspensions were added to ice-cold tubes containing 1 g of zirconium glass beads, 0.4 ml of phenol, 100 μl of chloroform, 30 μl of 10% sodium dodecyl sulfate, and 30 μl of 3 M sodium acetate (pH 5.2). The cells were disrupted with two 40-s treatments in a Fastprep (Qbiogene Inc., Illkirch, France) separated by 1 min on ice. After centrifugation, 200 μl of the aqueous phase was used for RNA isolation with a High Pure kit, which included 1 h of treatment with DNase I (Roche Diagnostics, Mannheim, Germany).

For detection of in vivo mRNA levels in an animal model, a mouse experiment was performed in an accredited establishment (no. A59107) according to the N°86/609/CEE guidelines of the French government. Seven-week-old female BALB/c mice were purchased from Iffa Credo (St. Germain sur l'Arbresle, France) and had free access to tap water and standard mouse chow during the experiments. After overnight culturing, bacterial cells were pelleted by centrifugation and resuspended at a concentration of 10¹⁰ CFU per ml in MRS. A mouse received a 100-μl (10⁹-CFU) dose of a freshly prepared bacterial suspension by intragastric administration, and the next day the mouse received a dose of 10¹⁰ CFU. Four hours later, the mouse was sacrificed, and a section of the mouse small intestine representing the duodenum (0.48 g) was quickly collected and frozen in liquid nitrogen until it was processed with a liquid N₂-cooled BioPulverizer (BioSpec Products, Bartlesville, Okla.). The powdered sample was immediately used for cell disruption and RNA isolation essentially as described above for pure bacterial cell pellets.

cDNA synthesis and quantitative reverse transcription-PCR.

The expression levels of L. plantarum WCFS1 genes derived from cells grown on MRS plates were compared to the expression levels of genes derived from cells grown on MRS plates containing 0.1% porcine bile and to the expression levels in the mouse duodenum. First, cDNA was synthesized by using Superscript III reverse transcriptase (Invitrogen, Breda, The Netherlands), 2 pmol of a gene-specific primer (Table 1), 40 U of RNaseOUT RNase inhibitor, each deoxynucleoside triphosphate at a concentration of 0.5 mM, and either 0.11 μg of total RNA from laboratory medium-grown L. plantarum WCFS1 or 2.1 μg of total RNA from a mouse GI tract sample. Reverse transcription was performed at 55°C for 60 min, and this was followed by inactivation of the reverse transcriptase by incubation at 70°C for 15 min. Primers were designed by using Primer 3 (www.genome.wi.mit.edu) and the software package Primer Express (PE Applied Biosystems, Nieuwekerk a/d Ijssel, The Netherlands). All primers were designed to have a melting temperature of 58 to 60°C, and the amplicon sizes ranged from 70 to 81 bp. Quantitative PCR was performed with the synthesized cDNAs by using an ABI Prism 7700 with SYBR Green technology (PE Applied Biosystems). Each 50-μl reaction mixture contained 1× SYBR Green master mixture (Applied Biosystems), each primer at a concentration of 400 nM (Table 1), and 0.1 or 200 ng of reverse-transcribed RNA from either plate-grown WCFS1 cells or a mouse GI tract sample. Amplification was initiated at 95°C for 10 min, and this was followed by 40 cycles of 95°C for 15 s and 55°C for 60 s. The identities of the amplicons resulting from the reactions with cDNA originating from culture- and mouse-derived templates were checked after amplification by melting curve analysis and amplicon DNA sequence analysis. Reaction mixtures containing no template and reaction mixtures containing DNase-treated RNA were included in each real-time PCR experiment to assess contamination and residual chromosomal DNA, respectively. Cycle threshold (C_t) values were obtained by manually setting the baseline and threshold values at which fluorescence was appreciably above the background fluorescence for each reaction in the exponential phase of amplification for all reactions. Relative transcript levels were calculated by using the comparative ΔΔC_t method described by Pfaffl et al. (24, 25). By using this method, PCR efficiencies were calculated with the equation E = 10^(−1/slope), where the slope is calculated from a standard curve of C_t values obtained for a dilution range of template cDNA. The average C_t values observed for the target gene transcripts (lp_0237, lp_0775, lp_1027, and lp_1898) were normalized to the average C_t values obtained for the reference gene transcripts (16S rRNA) from the same RNA sample. Three or four replicates of all samples and primer pairs were included in each quantitative PCR experiment, and all experiments were performed in triplicate. Statistical analyses of the differences in expression between samples were performed by using group means for statistical significance by the pairwise fixed reallocation randomization test, which was performed with the relative expression software tool (REST) (24, 25).

RESULTS

Growth characteristics and morphology of L. plantarum in the presence of bile salts.

The effects of different concentrations of bile salts on the morphology and growth of L. plantarum were investigated. After pregrowth in liquid MRS, L. plantarum WCFS1 was subcultured in MRS containing 0, 0.01, 0.05, 0.1, and 0.15% porcine bile salts. Growth was monitored for 24 h by measuring the optical density at 600 nm (OD₆₀₀), and the data were used to calculate the maximal growth rate of L. plantarum in the presence of different concentrations of bile salts (Fig. 1). The maximal growth rate was found to decrease significantly as the bile concentration increased. Moreover, the final OD₆₀₀ reached by L. plantarum grown in medium containing 0.1% porcine bile was approximately threefold lower than the final OD₆₀₀ reached by L. plantarum grown in standard MRS (data not shown). Four hours after inoculation logarithmically growing cells were collected from the cultures with different bile salt concentrations (Fig. 1), and the L. plantarum cell morphology was investigated by scanning electron microscopy (Fig. 2). Cells grown under standard conditions exhibited the characteristic rod-shaped, smooth-surface morphology of L. plantarum. When 0.05% bile was present in the medium, the cells had a slight tendency to clump together, their surfaces appeared to be less smooth, and membrane vesicle structures were visible. At bile concentrations of 0.1 and 0.15% these changes in morphology were more pronounced. Moreover, at these higher concentrations of bile a proportion of the bacterial cells had a shrunken and empty appearance. Similar observations were made when the growth rate and morphology on solid medium were investigated. Growth of colonies on MRS plates containing 0.1% bile salts was slightly retarded, and the colonies appeared to be very flat compared to the colonies on plates without added bile salts (data not shown). Since growth was the primary selection criterion in the conditional alr complementation screening analysis described below, it was essential to perform this screening analysis by comparing conditions at which the growth rates did not differ greatly. Therefore, a porcine bile concentration of 0.1% was chosen for identification of bile-inducible promoter elements in L. plantarum. Notably, 0.1% bile is in the physiological concentration range that occurs in the GI tract (2, 8).

FIG. 1.

Maximal growth rateS of L. plantarum in the presence of increasing concentrations of bile salts. A full-grown culture was diluted 50-fold in MRS containing no porcine bile (□), 0.01% porcine bile (▵), 0.05% porcine bile (○), 0.10% porcine bile (▪), or 0.15% porcine bile (▴), and growth was monitored by measuring the OD₆₀₀. The resulting data were used to calculate the maximal logarithmic growth rateS (0.56, 0.54, 0.52, 0.44, and 0.14 h⁻¹, respectively). The arrow indicates the time at which the morphology of the cells was investigated (Fig. 2).

FIG. 2.

Morphological changes in L. plantarum during bile salt stress. After 4 h of exposure to no bile salts (A), 0.05% bile salts (B), 0.10% bile salts (C), or 0.15% bile salts (D) cells were investigated by scanning electron microscopy.

Identification of bile-inducible genes by using the alr complementation library.

Use of the essential alanine racemase-encoding alr gene as a promoter probe in L. plantarum WCFS1 was described recently (5); this included analysis of a chromosomal L. plantarum WCFS1 library in the alr promoter probe vector pNZ7120. Here, the same library was screened for pNZ7120 derivatives harboring chromosomal L. plantarum WCFS1 fragments that contained promoter elements conditionally activated by 0.1% porcine bile. For 72 h the growth characteristics of approximately 4,000 colonies from the alr complementation library were compared on plates with and without bile, which resulted in the initial identification of 96 (2.4%) colonies displaying conditional growth only in the presence of bile. This conditional growth phenotype could be confirmed for 46 of these colonies when growth with and without bile was monitored for 72 h on plates containing d-cycloserine at concentrations ranging from 0 to 200 μg/ml. The partial sequences of the chromosomal inserts present in the pNZ7120 derivatives originating from 41 of the 46 clones were successfully determined. These sequences corresponded to 30 unique loci of the L. plantarum genome, since one locus was found six times, one locus was found three times, and four loci were found twice. Two independent clones (BI21 and BI49) both contained a different chromosomal fragment corresponding to lp_3415, encoding a transcription regulator homologue, and its upstream sequence (Table 2). Hence, the bile induction of lp_3415 was independently confirmed twice during the screening procedure. According to the current genome annotation database for L. plantarum WCFS1 (19), the loci harbor 31 unique genes and their upstream sequences in the proper orientation, which explains the observed induction of alr expression (Table 2). Notably, eight loci contained more than one putative 5′ end of an annotated open reading frame (ORF) and the upstream region. All ORFs that were identified in this alr complementation screening analysis were functionally categorized in groups involved in cell membrane function (eight ORFs), cell wall function (three ORFs), redox reactions (five ORFs), regulation (five ORFs), and other functions (four ORFs). The remaining six genes encoded (conserved) hypothetical proteins with unknown functions (Table 2).

TABLE 2.

Identification of clones in the alr complementation library that display conditional growth only in the presence of 0.1% porcine bile

Clone(s)	Insert start coordinate	Estimated insert size (kb)	lp no. (gene)a	Gene product	Growth with d-cycloserine at the following concn (μg/ml)b:
					0	2.5	5	10	25	50	100	200
BI1	79159	1.0	lp_0082c	Oxidoreductase			X	X
BI84	80981	2.3	lp_0085d	Cation efflux protein							X
BI19	215774	1.5	lp_0237d	Integral membrane protein						X	X	X
			lp_0240e	Hypothetical protein
BI79	711316	2.0	lp_0775 (argG)f	Argininosuccinate synthase						X	X
			lp_0774 (luxS)f	Autoinducer production protein
BI26, BI81	800854	2.0	lp_0858c	Redox protein, regulation of disulfide bond formation					X	X	X
BI65	1057803	1.5	lp_1158 (lys)g	Lysozyme					X	X	X
			lp_1157h	Transcription regulator (RpiR family)
BI87	1315054	0.9	lp_1435d	Integral membrane protein						X	X	X
BI14, BI95	1335147	1.5	lp_1459e	Conserved hypothetical protein				X	X
BI55	2013511	1.5	lp_2230e	Conserved hypothetical protein	X	X	X	X	X
BI9	2218907	1.5	lp_2484h	Transcription regulator (MarR family)		X	X	X	X	X
BI96	2284309	1.5	lp_2564d	Integral membrane protein					X	X	X	X
BI31	2528088	1.5	lp_2835f	2-Haloacid dehalogenase					X	X
BI64	2814574	2.0	lp_3145c	Oxidoreductase, N-terminal fragment					X	X
BI40	2819823	1.5	lp_3155d	Cell surface protein, ErfK family
			lp_3154 (acm3-C)g	Muramidase, C-terminal fragment						X	X
			lp_3153 (acm3-M)g	Muramidase, middle fragment
BI41	2821528	1.7	lp_3158c	Oxidoreductase
			lp_3159 (baiE)f	Bile acid 7α-dehydratase					X	X
			lp_3160d	Multidrug transport protein
BI28	2832336	2.0	lp_3175d	Integral membrane protein		X	X	X	X	X
BI45	2962988	2.0	lp_3330e	Conserved hypothetical protein					X	X
BI50	2976142	2.0	lp_3343e	Conserved hypothetical protein
			lp_3344h	Transcription regulator (MarR family)					X	X	X
			lp_3345 (nrpR4)h	Negative regulator of proteolysis
BI21, BI49	3032470	1.5	lp_3415e	Conserved hypothetical protein							X	X
BI29	3100458	1.5	lp_3488 (galR2)h	Galactose operon repressor (LacI family)							X	X
			lp_3489c	Oxidoreductase
BI2, BI7, BI8, BI14, BI63, BI69	3240643	1.5	lp_3626d	Sugar transport protein				X	X	X	X

^aWhen multiple ORFs in the correct orientation to drive alr expression are present in a clone, the ORF that is located closest to the alr gene is presented first.

^bAn X indicates that growth was observed only in the presence of bile.

^cRedox reaction gene.

^dGene with a membrane-associated function.

^eGene encoding a hypothetical protein.

^fGene with another function.

^gGene with a cell-wall-associated function.

^hRegulation gene.

Clone BI41 harbors three genes, including a gene encoding a homologue of the bile acid 7α-dehydratase of Eubacterium sp. strain VPI 12708 and Clostridium sp. strain TO-931 (30 and 28% identity at the protein level, respectively) (21, 36). In these intestinal organisms the baiE gene, encoding bile acid 7α-dehydratase, is localized in an operon. In Eubacterium the expression of this operon appeared to be induced in the presence of bile (21). A similar operon structure is not found in L. plantarum. Nevertheless, the fact that this baiE homologue was identified in the alr complementation screening analysis strongly suggests that the expression of this single gene is also regulated by bile in L. plantarum WCFS1. Clone BI87 harbors a 3′-truncated fragment of lp_1435, encoding an integral membrane protein, which was previously identified in an alr complementation screening analysis for high-salt-inducible promoters of L. plantarum (5). These findings suggest that there is a partial overlap in the responses of this organism to high NaCl concentrations and bile salts, possibly caused by the membrane stress induced under both these conditions. A very striking observation was the identification of lp_0237 and lp_0775, encoding an integral membrane protein and an argininosuccinate synthase, in clones BI19 and BI79, respectively (Table 2). Both of these genes were previously identified in our laboratory by a resolvase-based in vivo expression technology approach as being important for L. plantarum during passage through the GI tract of mice (4).

Expression analysis of lp_0237 and lp_0775.

The bile-inducible characteristics of lp_0237 and lp_0775 were investigated further by quantitative reverse transcription-PCR. RNA was isolated from L. plantarum cells grown on plates with and without bile. The isolated RNA samples were used for gene-specific synthesis of cDNA, which was used as a template for quantitative real-time PCR with specific primers for the bile-induced genes lp_0237 and lp_0775 and the 16S rRNA gene. The latter RNA was used to correct for the total amount of L. plantarum-specific RNA added to the different reaction mixtures. Negative control reaction mixtures containing the L. plantarum-specific16S rRNA primers and DNase-treated RNA were included in each PCR. These reactions never produced any detectable amplicon, indicating that there was no DNA contamination in the RNA samples. Moreover, the specificity of the PCRs was confirmed by a combination of melting curve analysis and DNA sequence analysis of the amplicons (data not shown). All signals were correlated to the 16S rRNA signal derived from the corresponding cDNA samples. The in vitro induction of lp_0237 and lp_0775 by the presence of porcine bile was investigated. This experiment showed that the expression levels of lp_0237 and lp_0775 were significantly induced (24- and 4-fold, respectively) in cells grown on plates containing 0.1% porcine bile compared to control plates lacking bile (Table 3). These data demonstrate that the bile-inducible regulatory characteristics obtained for these two genes by using the plasmid-based alr promoter probe can be extrapolated to the native, single-copy situation on the chromosome. Moreover, the observed induction by bile suggests that the previously observed in vivo induction (4) of lp_0237 and lp_0775 occurs in the duodenum, as this is the site of bile release in the host. Therefore, RNA was isolated from duodenum samples from a mouse fed L. plantarum, and a second quantitative reverse transcriptase PCR experiment was performed to assess the in vivo expression levels of lp_0237 and lp_0775 in the duodenum. This experiment revealed significantly higher expression levels (13- and 29-fold, respectively) for these genes during passage through the mouse duodenum than in L. plantarum grown on MRS, while in an identical experiment the expression levels of two L. plantarum household genes (lp_1027 [fusA2] encoding an elongation factor and lp_1898 [pfk] encoding 6-phosphofructokinase) were not significantly increased in vivo compared to the expression levels in MRS (Table 3). These data demonstrate that the in vitro regulatory characteristics observed for lp_0237 and lp_0775 can be translated to the in vivo situation in the mouse duodenum and strongly suggest that contact with bile is the inducing environmental factor in vivo.

TABLE 3.

Levels of induction of L. plantarum genes in vitro by 0.1% bile and in situ in the duodenum of a mouse model compared to the level in MRS without bile

lp no. (gene)	Function	Fold induction ina:
		MRS + 0.1% bile	Duodenum
lp_0237	Integral membrane protein	23.7b	12.5b
lp_0775 (argG)	Argininosuccinate synthase	4.3b	28.9b
lp_1027 (fusA2)	Elongation factor	NDc	2.5
lp_1898 (pfk)	6-Phosphofructokinase	ND	1.4

^aThe coefficient of variation among replicates (n = 3) was <25%.

^bThe relative expression level of the target gene is significantly different from the level observed in MRS lacking bile according to the pairwise fixed reallocation randomization test (P < 0.05).

^cND, not determined.

DISCUSSION

In this paper we describe the growth, morphological, and genetic responses of L. plantarum WCFS1 to bile salts. A stepwise increase in the porcine bile concentration resulted in a stepwise decrease in the maximal growth rate and the final OD₆₀₀ of L. plantarum. The observed gradual decrease in the growth rate coincided with the gradually increasing severity of changes in morphology, including bulky structures on the cell surface, the formation of membrane vesicles, and clumping of the cells. Moreover, the observed formation of ghost cells suggests that cell wall integrity was lost after addition of bile, possibly leading to leakage of intracellular material from the cells and a disturbed energy balance. The bile-induced morphological changes in L. plantarum are very similar to those observed in P. freudenreichii (20). Furthermore, leakage of proteins from cells after bile treatment was previously observed in other LAB, including P. freudenreichii and Lactobacillus acidophilus (20, 22). Since a porcine bile concentration of 0.1% had only a marginal effect on the growth rate but nevertheless resulted in severe morphological changes, this physiologically relevant concentration was used in the rest of the experiments.

The previously constructed alr complementation library (5) was exploited for identification of clones containing L. plantarum chromosomal fragments that harbor promoter elements conditionally activated by bile. This approach resulted in the identification of 30 unique loci harboring 31 putative genes whose expression is potentially induced by bile. The putative genes identified as bile inducible were organized in six functional categories (Table 2). Strikingly, 11 of these ORFs encode proteins involved in membrane- and cell wall-associated functions. The induction of this relatively high number of genes involved in cell envelope functions is in agreement with the observed morphological changes in L. plantarum in the presence of bile. Notably, genes encoding putative functions involved in fatty acid and cell wall biosynthesis have previously been identified as important for the bile resistance of E. faecalis (3). The group of genes encoding membrane-associated functions includes the genes encoding three possible exporter proteins, namely, lp_0085, lp_2564, and lp_3160. The latter gene is annotated as a multidrug transporter gene, suggesting a possible role in the export of bile or bile-derived compounds. Similarly, lp_2564, encoding a protein with significant homology to a permease in Bacillus cereus, could play a role in the export of bile. Finally, lp_0085, encoding a putative efflux protein, might be involved in maintenance of the electrochemical membrane potential under bile-induced stress conditions. Remarkably, the importance of efflux pumps in bile resistance was previously demonstrated in several bacteria, including Escherichia coli and L. monocytogenes (2, 15). The genes encoding three cell wall-associated functions identified here include a putative lysozyme gene and two genes annotated to encode fragments of a possible muramidase. Moreover, BlastP analysis of lp_3154 demonstrated that there was 35% identity with the gene encoding a choline binding protein from Streptococcus pneumoniae. The chemical structure of choline is similar to that of bile. Therefore, the protein encoded by lp_3154 could be important in the defense of L. plantarum against bile salts.

Five genes identified here as bile-inducible genes are annotated as having functions involved in redox reactions (namely, four oxidoreductases and a redox protein acting as a regulator of disulfide bond formation), suggesting that bile-induced redox balance disturbance and/or oxidative stress occurs. Notably, a gene encoding a function involved in a redox reaction was previously recognized as important during bile stress in E. faecalis (3). Another group of five genes encoding regulatory functions was identified as bile inducible in L. plantarum. Remarkably, two of these genes (lp_2484 and lp_3344) belong to the marR family of regulators. In several bacteria, including E. coli and Salmonella enterica serovar Typhimurium, the MarA and MarR proteins mediate the expression of a diverse set of genes involved in multidrug resistance, including genes encoding multidrug efflux proteins (14, 32). The lp_3344 gene product exhibits 28% identity with MarR from S. enterica serovar Typhimurium. Moreover, the conserved hypothetical protein encoded by lp_3415 exhibits 30% identity with MarA from S. enterica serovar Typhimurium. Therefore, lp_3344 and lp_3415 might be involved in the regulation of multidrug transporters in L. plantarum, possibly including the two transporters encoded by lp_2564 and lp_3160 mentioned above (Table 2). Next to lp_3415 four other conserved genes and one unique hypothetical protein gene were identified in the screening for bile-inducible L. plantarum ORFs. The role of these genes in bile resistance remains to be determined.

Bile induction could reflect the in situ conditions encountered by L. plantarum during passage through the gastrointestinal tract. Two striking findings in this context are lp_0237 and lp_0775, which we identified as bile-inducible genes in the in vitro alr complementation screening analysis described here; these genes have been identified previously in L. plantarum as genes that are induced in vivo during passage through the mouse GI tract (4). By using quantitative reverse transcription-PCR, the in vitro induction by bile and the in vivo induction in the duodenum of a mouse model system compared to laboratory conditions could be established for these genes. In conclusion, in this paper we provide valuable data on the in vitro genetic response of L. plantarum to bile. The experiments described here demonstrated that simplified in vitro mimicking of complex environmental niches can result in the identification of genes that are relevant in situ in these niches. Moreover, this approach potentially provides clues to the environmental trigger involved in the in situ regulation of specific genes, which should enable future unraveling of the genetic behavior of L. plantarum during passage through (specific parts of) the GI tract.