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Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk

Department of Food, Bioprocessing, and Nutrition Sciences, and Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695

³ Corresponding author: klaenhammer{at}ncsu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus NCFM cells propagated in 11% skim milk during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 open reading frames were differentially expressed at least in one time point. Genes differentially expressed in skim milk included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4 h, whereas gene expression of Opp2 increased over time reaching its highest level at 12 h, suggesting that the 2 systems have different specificities. Expression of a 2-component regulatory system, previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (autoinducer-2) and adherence to mucin and intestinal epithelial cells, in vitro.

Key Words: Lactobacillus acidophilus • probiotic • microarray analysis • dairy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Lactic acid bacteria (LAB) constitute a varied group of microorganisms that occur naturally in dairy, meat, and plants (cabbage, corn, barley, mashes, kale, and silage; Carr et al., 2002). The most important application of LAB has been their use as starter cultures in the manufacture of fermented foods, and notably dairy products such as cheese, yogurt, and fermented milk. Today, the dominant vehicles for delivery of probiotic strains in foods are yogurt and fermented milk. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt (Sanders and Klaenhammer, 2001), with approximately 80% of the yogurt in the United States now containing L. acidophilus (Hutkins, 2006). Although this microorganism does not participate in the actual yogurt fermentation, it grows slowly in milk during product manufacture (Shah, 2000). Consequently, it is a common practice to incorporate yogurt cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) along with probiotic cultures (L. acidophilus, Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium spp.) in many yogurts and fermented milks (Shah, 2000).

Probiotics are "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (Reid et al., 2003). The International Dairy Federation recommends that dairy products contain at least 10⁷ cfu/mL (Champagne et al., 2005). The actual number of colony-forming units that reach the distal portion of the gastrointestinal tract remains debatable, but clearly depends on several aspects that include acid and bile tolerance of the probiotic strain, manufacturing conditions, and survival of the microorganism during the fermentation and postfermentation (cooling and storage) stages. The major factor affecting viability and performance of probiotic strains during processing is the food matrix formulation (Mattila-Sandholm et al., 2002). Additionally, survival of probiotic strains during storage is a key issue in the food industry. Several studies (reviewed by Champagne et al., 2005) have reported that pH, oxygen, and starter production conditions have significant effects on survival and stability of probiotic cultures during storage in yogurt.

The selection criteria for probiotics include general safety and stability aspects and functional properties (Tuomola et al., 2001). To survive passage through the stomach and small intestine, probiotic strains must tolerate the acidic and digestive protease conditions of the stomach, and must survive and grow in the presence of bile acids. The ability to adhere to the mucosa and intestinal tissues has been related to retention in the gastrointestinal tract and immunomodulation (Grangette et al., 2005; Hisbergues et al., 2007). In this regard, L. acidophilus NCFM, delivered widely in yogurt and fermented milks, has been shown to adhere to enterocyte-like Caco-2 cells via cell surface proteins such as a fibronectin-binding protein, a mucin-binding protein, and a surface layer protein (Buck et al., 2005).

Milk is the dominant food vehicle and substrate for delivery of probiotic strains to humans. Our objective was to investigate how growth in milk affected gene expression in L. acidophilus NCFM and determine if any potential probiotic traits were affected during exposure to, and fermentation of, milk.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Strains, Plasmids, and Culture Conditions
The bacterial strains used in this study are listed in Table 1. Escherichia coli were propagated at 37°C in Luria-Bertani broth (Difco Laboratories Inc., Detroit, MI) with shaking. Escherichia coli cultures were propagated on brain heart infusion agar (Difco), supplemented with 150 µg of erythromycin/mL, when appropriate. Lactobacilli were propagated statically at 37°C in de Man, Rogosa, and Sharpe broth (MRS; Difco), or MRS agar supplemented with 1.5% agar. When appropriate, erythromycin (5.0 µg/mL) or chloramphenicol (4.0 µg/mL) was added to MRS broth or agar plates. For growth and microarray experiments, L. acidophilus was propagated at 37°C in 11% reconstituted skim milk (SM; MP Biomedicals Inc., Solon, OH). After reconstitution, the milk was steamed at 100°C for 40 min and immediately placed in an ice bath.

Microarray Experimental Design and RNA Isolation
A loop experimental design (Vinciotti et al., 2005) was employed in a time course experiment to monitor the transcriptional response of L. acidophilus NCFM during growth in 11% reconstituted skim milk. The culture was initiated in MRS broth and transferred 3 to 5 times in SM before a 10% inoculation in prewarmed SM. Cells and RNA samples were obtained from 2 biological replicates at times of 5 min (pH = 6.4; 2.1 x 10⁷ cfu/mL), 4 h (pH = 5.8; 3.8 x 10⁷ cfu/mL), 8 h (pH = 5.4; 7.1 x 10⁷ cfu/mL), and 12 h (pH = 4.9; 1.5 x 10⁸ cfu/mL; see Supplementary Figure 1 available online: http://jds.fass.org/content/vol92/issue3/) and compared in an unbalanced loop-design. Samples were compared as follows (by dye used, Cy3 versus Cy5; Amersham Biosciences, Piscataway, NJ): T1 (5 min) versus T2 (4 h), T2 versus T3 (8 h), T3 versus T4 (12 h), T4 versus T2, and T3 versus T1. One-hundred-milliliter aliquots of L. acidophilus cells growing in SM were harvested by centrifugation for 10 min at 3,220 x g, and frozen immediately in a dry ice/ethanol bath (the total time of each sampling process was 5 min). Isolation of RNA was performed as described previously (Azcarate-Peril et al., 2005) with the following modifications: 5 mL of Trizol reagent (Invitrogen, Carlsbad, CA) was added to the cell pellets and they were homogenized in a Mini-Beadbeater-8 cell disruptor (Biospec Products, Bartlesville, OK) for seven 40-s cycles (and chilled on ice for 40 s between cycles); the phases were then separated by centrifugation (3,220 x g for 20 min at 4°C). The aqueous phase was removed to a fresh tube and 2.1 mL of Trizol and 1 mL of chloroform were added. The mixture was vortexed for 15 s and centrifuged (3,220 x g for 15 min at 4°C) to separate the phases. The Trizol step was repeated twice and RNA was precipitated from the final aqueous phase by adding 1 volume of isopropanol, followed by incubation at room temperature for 10 min and centrifugation (11,600 x g for 10 min at 4°C). Purity and quality of RNA samples were determined by electrophoresis on agarose gels. NanoDrop (ND-3300, NanoDrop Technologies, Wilmington, DE) was used as an ultraviolet/visual spectrophotometer for quantification of the RNA samples.

Microarray Hybridization
A previously developed and validated microarray platform based on the PCR products of predicted ORF from L. acidophilus NCFM (Azcarate-Peril et al., 2005) was used to monitor changes in gene expression. Reverse transcription and labeling of identical amounts (20 µg) of DNase-treated (Invitrogen) RNA was performed with the SuperScript Indirect cDNA Labeling System for DNA Microarrays (Invitrogen) according to the manufacturer’s directions. Coupling of the Cy3 and Cy5 dyes to the aa-dUTP (2'-deoxyuridine 5'-triphosphate) labeled cDNA and hybridization of samples to microarrays were performed according to the protocols outlined by Hegde et al. (2000) and described previously (Azcarate-Peril et al., 2005).

Data Preprocessing and Analysis
After acquisition of fluorescence intensities at 10-µm resolution using a ScanArray 4000 Microarray Scanner (Packard Biochip BioScience; Biochip Technologies LLC, Billerica, MA), signal intensities (stored as TIFF files) were quantified, and the local background was subtracted using the QuantArray 3.0 software package (Packard BioScience). Afterwards, data were log₂ transformed and imported into JMP Genomics (SAS, Cary, NC). Loess normalization was applied to preprocessed data. Following normalization, gene-specific effects were modeled in terms of the residuals. A mixed model analysis was applied to the prenormalized data (Cui and Churchill, 2003). False discovery rate was selected to adjust for the large number of multiple hypothesis tests across all genes. The default value of (0.05) was used. The model-based approach, step-down quadratic regression method was applied to 791 significant genes for pattern recognition (Liu et al., 2005).

Microarray Data Submission
Microarray platform and data are available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession numbers GPL1401 (platform) and GSE12924 (series).

Real-Time Quantitative PCR
Real-time quantitative (qRT) PCR was used to confirm microarray results. The protocols described previously (Azcarate-Peril et al., 2006) were used for amplification, quantification, and normalization of PCR products. The same RNA samples employed for microarray hybridizations were used for qRT-PCR. The RNA was DNase treated, quantified, and diluted to a concentration of 50 ng/µL. The absence of contaminating genomic DNA was confirmed by PCR of the DNase-treated samples. Reverse transcription and PCR were carried out with an iCycler iQ (BioRad Laboratories, Hercules, CA) using the one-step Quanti-Tect SYBR Green RT-PCR kit (Qiagen, Valencia, CA). Primers meeting the standard criteria for qRT-PCR for the following genes were designed using CloneManager (Scientific & Educational Software, Cary, NC) software (Supplementary Table 1 available online: http://jds.fass.org/content/vol92/issue3/): LBA0367 (encoding a putative receptor), LBA0395 (formyl-CoA transferase), LBA0396 (oxalyl-CoA decarboxylase), LBA0555 (myosin-crossreactive antigen), LBA1234 (cadmium/manganese transport ATPase), LBA1249 (heat inducible transcription repressor HrcA), LBA1512 (PrtP precursor), and LBA1588 (PrtM precursor).

The induction ratios measured by microarrays were plotted against induction ratios measured by qRT-PCR, for the 8 selected genes and 4 time points, to validate microarray data (Supplementary Table 2 and Supplementary Figure 2 available online: http://jds.fass.org/content/vol92/issue3/). Individual r values ranged between 0.44 and 0.99 for each of the tested genes with an overall r value of 0.86. As reported previously, (Azcarate-Peril et al., 2006), qRT-PCR measurements revealed larger induction levels.

Acidification Activity of L. acidophilus Cultures in Milk
The acidification capability of L. acidophilus cultures was measured using the Orion EA940 Multi-Channel Benchtop Meter and Multi-Channel Electrode Switch-box (Thermo Fisher Scientific Inc., Waltham, MA). Lactobacillus acidophilus cultures grown overnight in MRS broth were centrifuged (3,220 x g for 10 min at room temperature). The pellet was washed twice with phosphate buffer and resuspended in the same buffer. Forty milliliters of prewarmed SM was inoculated with 0.5 to 2.2 x 10⁷ cells. Acidification curves were performed at 37°C in 11% SM. Four independent experiments were performed for each strain and the acidification rate was calculated as the first time derivative. The time in minutes necessary to reach the maximum acidification rate in milk (tm) was calculated as described by Fonseca et al. (2000). The tm values were correlated to the natural logarithm of the initial bacterial population (in cfu/mL) as described by Fonseca et al. (2000).

Survival of L. acidophilus Cultures in Acidified Milk
Survival of L. acidophilus cultures in SM acidified at pH 4.5 (with lactic acid) at 4°C was examined as follows. Lactobacillus acidophilus cultures grown overnight in MRS broth at 37°C were centrifuged (3,220 x g for 10 min at room temperature) and the pellet washed twice with phosphate buffer and resuspended in the same buffer. Fifty milliliters of SM adjusted to pH 4.5 with lactic acid were inoculated with 1.8 to 4.1 x 10⁸ cells. Survival curves were performed at 4°C. Viable-cell counts were determined at 24- to 48-h intervals by plating serial dilutions in a 10% MRS broth diluent onto MRS agar using a Whitley Automatic Spiral Plater (Don Whitley Scientific Limited, West Yorkshire, England).

Adhesion of L. acidophilus to Mucin
Mucin and Plate Preparation.
The adhesion ability of L. acidophilus derivatives was examined by the method of Vishwanath and Ramphal (1984) with minor modifications. One hundred microliters of a 10 mg/mL solution of partially purified type III porcine gastric mucin (Sigma Chemical Co., St. Louis, MO) were immobilized in polystyrene 96-well microtiter plates (Corning Inc., Acton, MA) by overnight incubation at 4°C. Any surplus of mucin was removed by pipetting and wells were washed twice with 200 µL of PBS. Adhesion experiments were carried out 3 times, each time in triplicate.

Preparation of Microorganisms for Adhesion Assay.
Lactobacillus acidophilus cultures were grown in SM at 37°C until pH 6.0 was reached. One hundred microliters of the bacterial suspension was added to each well and the plates were incubated 1 h at 37°C. Each well was washed 7 times with 200 µL of sterile PBS (to remove unbound bacteria), then treated with 200 µL of a 0.05% (vol/vol) Triton X-100 solution (to desorb bound bacteria). One hundred microliters of the contents of each well were removed, diluted in PBS, and plated on MRS agar plates. The viability of L. acidophilus strains was not affected by Triton X-100 under the conditions used (data not shown). Three wells per sample were used and each adherence assay was conducted in triplicate.

Adhesion to Caco-2 Cells
The adherence of Lactobacillus strains to epithelial cells was carried out as described by Buck et al. (2005) with the following modifications: Caco-2 cells from passage 24 to 29 were routinely grown in Dulbecco modified Eagle’s minimal essential medium (DMEM) supplemented with 20% inactivated fetal bovine serum, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 mg/mL penicillin G, 100 mg/mL streptomycin sulfate and 0.25 mg/mL amphotericin (Gibco, Invitrogen, Carlsbad, CA). Monolayers of Caco-2 cells were seeded at a concentration of 6.5 x 10⁴ cells per well in 12-well tissue plates, and incubated at 37°C in a 95% air-5% CO₂ atmosphere. Cells were used at late postconfluence culture after 21 d, with a change of medium every 2 d. The monolayer was washed twice with 1 mL of PBS to remove traces of antibiotics, and 1 mL of Dulbecco’s modified Eagle’s medium without antibiotics was added to each well. The plates were incubated for 1 h at 37°C before the adherence assay.

Lactobacillus acidophilus cultures were grown at 37°C until pH 6.0 in 15 mL of either MRS or SM, inoculated with 750 µL of an overnight culture. To study the influence of the growth medium on adhesion of L. acidophilus NCFM, cultures in MRS or SM were centrifuged at 3,220 x g for 10 min, and the pellets were washed once with an equal volume of phosphate buffer (pH 6.0) and resuspended either in phosphate buffer (pH 6.0) or SM acidified to pH 6.0 with lactic acid. Initial cell counts per milliliter were determined in both samples to ensure comparable counts between samples to be used in adherence assays. To compare the adhesion ability of selected L. acidophilus mutant strains (listed in Table 1), cultures were grown in SM until reaching pH 6.0, and then the cells were removed, resuspended, counted, and used directly in adhesion assays.

One milliliter of each sample was added to each well of the tissue culture plate and incubated at 37°C for 1 h. Monolayers were washed 5 times with 1 mL of PBS and cells were desorbed by addition of 1 mL of 0.05% Triton-X100. The number of viable adhering bacteria was determined by plating serial dilutions on MRS agar plates. Three wells per sample were used and each adherence assay was conducted in triplicate. Bacterial adhesion was expressed as a percentage of the control strain.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Growth and Acidification Activity
To determine the optimal culture conditions for preparation of cells for the microarray studies, the growth and acidification of L. acidophilus NCFM 37°C in MRS and 11% skim milk (SM) was examined, in triplicate (Supplementary Figure 1 available online: http://jds.fass.org/content/vol92/issue3/). The maximum specific growth rate (µ_max) of L. acidophilus in SM was less than half of the µ_max of the strain growing in MRS (0.17 and 0.47 per h, respectively). The acidification rate of L. acidophilus in MRS and SM was evaluated by continuous measurement of pH. No differences in acidification rates were observed between MRS and SM during the first 3 h of incubation, each decreasing the pH 0.05 units per h). At 9 h, L. acidophilus NCFM decreased the culture pH by 0.14 and 0.055 units per h for MRS and SM, respectively.

Gene Expression Profile of L. acidophilus During Growth in Milk
A loop experimental design was employed in a time course experiment to monitor the transcriptional response of L. acidophilus NCFM during growth in SM. We obtained RNA at times 5 min (pH = 6.4; 2.1 x 10⁷ cfu/mL), 4 h (pH = 5.8; 3.8 x 10⁷ cfu/mL), 8 h (pH = 5.4; 7.1 x 10⁷ cfu/mL), and 12 h (pH = 4.9; 1.5 x 10⁸ cfu/mL; see Supplementary Figure 1 available online: http://jds.fass.org/content/vol92/issue3/) and the RNA was compared in an unbalanced loop-design. The statistical filter [–log10 (P-values) < –1.98] allowed 791 genes to pass, with 398 that were differentially expressed between paired time points in at least one time point, representing 21% of 1,864 ORF in the complete genome of L. acidophilus.

A clusters of orthologous groups (COG) classification of differentially expressed genes during growth in SM (Figure 1) indicated that the majority of genes induced at 4 h belonged to the translation, ribosomal structure and biogenesis [J] category, whereas most of the under-expressed genes belonged to carbohydrate transport and metabolism [G]. At 8 h, amino acid transport and metabolism [E] and [J] were the most up-regulated categories, and the majority of down-regulated genes were either unknown or could not be included in any COG category. Interestingly, at 12 h most of up-regulated genes were in an unknown/no GOG category, followed by genes included in the categories [E], [J], and cell wall, membrane, envelope biogenesis [M]. Again, the majority of genes down-regulated was in the unknown/no GOG category or included in [G]. However, the third most represented category was replication, recombination and repair [L].

View larger version (33K): [in this window] [in a new window]   Figure 1. Temporal clusters of orthologous groups (COG; Tatusov et al., 1997) classification of differentially expressed genes at 4, 8, and 12 h. Genes were classified according to the main COG domain present in the potentially encoded protein sequence. The COG functional category codes are indicated on the right.

View larger version (23K): [in this window] [in a new window]   Figure 2. Temporal expression patterns of genes of the proteolytic system identified by the quadratic regression method (Liu et al., 2005). Patterns obtained included: Flat (not shown; no significant differential expression over time), linear up-regulated regression pattern (LU), quadratic concave regulated regression pattern (QC), quadratic convex regulated regression pattern (QV), quadratic linear concave up-regulated regression pattern (QLCU), and quadratic linear convex up-regulated regression pattern (QLVU).

Previously conducted computer simulation analysis of predicted transporters in the L. acidophilus genome revealed the presence of 2 complete oligopeptide ABC transporters (Altermann et al., 2005; Azcarate-Peril et al., 2005): Opp1 (ORF LBA0197 to LBA0203) and Opp2 (ORF LBA1300 to LBA1306). Each consists of 6 genes coding for 2 ATP-binding proteins (OppD and OppF), 2 membrane proteins (OppB and OppC), and 2 substrate-binding proteins (OppA and OppA-B). Expression of Opp1 fell into the QLCU temporal expression pattern. In fact, Opp1 reached its maximum expression level at 4 h. Expression of Opp2 increased over time falling into the LU pattern (Figure 3). These results suggest that Opp1 and Opp2 might have different specificities. In addition to the 4 periplasmic substrate-binding proteins (OppA) encoded within Opp operons, 6 additional putative oppA genes were identified in L. acidophilus (LBA1216, LBA1347, LBA1400, LBA1665, LBA1958, and LBA1961). Only 4 oppA genes were differentially expressed over time: LBA0197, LBA0198 (adjacent to Opp1), and LBA1301 (adjacent to Opp2) fell in the QC regression pattern, and LBA1665 was included in the LU pattern.

View larger version (15K): [in this window] [in a new window]   Figure 3. Mean gene expression of oligopeptide transport 1 (opp1, continuous line), opp2 (dashed line), and LBA1524HPK-LBA1525RR 2-component regulatory system (dotted line) over time during growth of L. acidophilus NCFM in milk.

Expression of Carbohydrate Utilization Genes
Lactose is the principal carbohydrate in the milks of all mammals. Milk also contains trace amounts of other sugars, including glucose, fructose, glucosamine, galactosamine, neuraminic acid, and small amounts of oligosaccharides, with sialyllactose being the most represented in bovine milk (Kunz et al., 2000). Lactobacillus acidophilus NCFM can transport and utilize glucose, sucrose, fructose, trehalose, melibiose, gentiobiose, cellobiose, maltose, mannose, fructooligosaccharides, raffinose, lactose, galactose, amygdalin, salicin, arbutin, and N-acetyl-glucosamine (Altermann et al., 2005; Barrangou et al., 2006). Several sugar transporters were differentially expressed in L. acidophilus growing in SM. Among the phosphotransferase transport systems, a lactose-specific transporter (LBA0989) was overexpressed at 12 h, 2 cellobiose-specific transporters (LBA0491 and LBA0606) were overexpressed at 4 h, and an arbutin-specific transporter was repressed at times 4 and 12 h. The ABC transporters responsible for the uptake of maltose (LBA1865–LBA1866), lactose/galactose (LBA1463) and one uncharacterized sugar transporter (LBA0045) were highly expressed at 5 min and under expressed at times 4, 8, and 12 h. Interestingly, an uncharacterized sugar transport system encoded by LBA1944 to LBA1946 was induced mainly during logarithmic growth phase, at 8 h. The genes/systems involved in carbohydrate utilization differentially expressed upon exposure to milk included lactose/galactose, fructose, maltose and N-acetyl-glucosamine. In contrast with the expression patterns of genes involved in proteolysis, expression of genes involved in lactose utilization were increased at time 5 min, suggesting that this region is rapidly induced upon exposure of L. acidophilus to the dairy environment, especially when cultures have previously been transferred in SM. The same pattern was observed for genes involved in maltose and fructose utilization. On the other hand, N-acetylglucosamine-6-P deacetylase (LBA0144) and glucosamine-6-phosphate isomerase (LBA1948), involved in the utilization of glucosamine, were among the highest genes induced at 4 h. In fact, microarray data indicated that L. acidophilus uses the glucosamine-6-phosphate isomerase (LBA1948; EC 3.5.99.6) and not the D-fructose-6-phosphate amido-transferase (LBA0462; EC 2.6.1.16) to process glucosamine 6-phosphate generating fructose 6-phosphate (and ammonia).

Components of the general carbohydrate utilization regulon were differentially expressed in L. acidophilus during growth in milk; specifically, ptsH (HPr, LBA0639) ptsI (EI, LBA0640), and ccpA (CcpA, LBA0431) showed an LU trend indicating increased expression over time

Stress, Quorum Sensing, and 2-Component Regulatory Systems
Step-down quadratic regression analysis (Liu et al., 2005) applied to statistically significant genes for pattern recognition indicated that 24 genes, potentially involved in the stress response of L. acidophilus, showed biologically meaningful temporal expression patterns (Table 3). Interestingly the gene responsible for the generation of the autoinducer-2 (AI2) molecule, which mediates cell-density responses, followed a QLVU pattern of expression. The genome sequence of L. acidophilus NCFM contains the complete pathway for the production of AI2 (Buck et al., 2008); however, only luxS (LBA1081) was differentially expressed.

Expression of Adhesion Genes in SM
Adhesion to the intestinal mucosa is considered an important trait for bacterial retention in the gastrointestinal tract and interaction with immunomodulatory cells of the mucosal immune system. Consequently, the capacity to adhere to the intestinal mucosa is a very important selection criterion for probiotics (Tuomola et al., 2001). The genome sequence of L. acidophilus indicated the presence of several genes encoding putative adhesion factors (Altermann et al., 2005), and some of these have already been shown to play a role in adherence to Caco-2 epithelial cells, in vitro (Buck et al., 2005). Genes putatively involved in adhesion, containing a cell wall anchoring motif LPxTG or with similarities to previously characterized adhesion genes, differentially expressed during growth of L. acidophilus in SM are listed in Table 4. Eight genes encoding proteins containing the LPxTG and/or LPQTxE motif were differentially expressed; however, only 3 of them (2 mub genes, LBA1392 and LBA1652, and thrS) showed recognizable patterns of expression over time. Expression of ORF LBA0493 and LBA1850, encoding putative aggregation-promoting proteins, increased over time falling into QLCU and QLVU temporal patterns, respectively. LBA0493 was one of the most induced genes over time [up to 5.6-fold (2.5-fold in log2 scale) at 4 h].

The method described by Fonseca et al. (2000) was used to correlate acidification rates with the initial cell concentration to determine if any of the inactivated genes were involved in acidification activity and, hence, growth of L. acidophilus in milk (Table 5). Four mutant strains showed significantly (P < 0.1) different tm values than their respective control strain. As expected, the histidine protein kinase (LBA1524HPK) mutant previously characterized as involved in the regulation of genes involved with acid resistance and proteolysis showed an increased tm value in SM (Azcarate-Peril et al., 2005). The other mutant strains showing significantly different tm values were cdpA (Altermann et al., 2004), mucin-binding protein (LBA1392; Buck et al., 2005), and an uncharacterized putative myosin cross-reactive antigen (LBA0555; A. Dobson, North Carolina State University; unpublished results).

View larger version (14K): [in this window] [in a new window]   Figure 4. Correlation between tm (time in minutes necessary to reach the maximum acidification rate in milk) and K (death rate) values corresponding to acidification rates in milk and survival in acidified milk, respectively.

To identify potential associations between genes differentially expressed in SM and adhesion properties, we compared the ability of L. acidophilus NCFM versus 6 selected mutants (see Table 1) to adhere to mucin and to Caco2-tissue culture cells. Two mutant strains, NCK1686 (NCFM::LBA1524HPK) and NCK1822 (NCFM::LBA1690) showed a 30% decrease in adhesion to Caco-2 cells (Figure 5), suggesting that these proteins may play a role in adhesion of L. acidophilus when grown in milk. On mucin, NCK1686 showed a 31% reduction and NCK1822 more than a 68% reduction in adhesion. None of the other mutant strains were affected in adhesion on the 2 models tested, indicating that the insertionally inactivated genes in these strains; that is, myosin-crossreactive antigen (LBA0555), mucus binding protein precursor (LBA1392), and the β-galactosidase large subunit (LBA1398), are not involved in adherence of L. acidophilus when grown in milk.

View larger version (13K): [in this window] [in a new window]   Figure 5. Adherence abilities of 6 L. acidophilus NCFM derivatives grown in skim milk, to mucin (), and to Caco-2 cells (). The results are expressed as a percentage of the wild-type strain. Values represent the mean of 3 independent experiments and error bars represent the standard deviation. The asterisks represent statistically significant (P < 0.05) differences between mutant and control strains.

Conversely, proteolytic genes showed increasing patterns of expression over time. Interestingly, the 2 oligopeptide operons (opp1 and opp2) encoded by L. acidophilus, which are involved in the transport of oligopeptides into the cell, showed different expression patterns. The maximum expression level of opp1 was at 4 h (early logarithmic phase of growth), but the expression of opp2 reached its maximum level at 12 h (early stationary phase). Expression of the 2-component regulatory LBA1524HPK-LBA1525RR in milk correlated with the expression of the oligopeptide transport systems as it showed increased levels of expression at 4 and 12 h (Figure 3). These results support previous data that indicated that LBA1524HPK-LBA1525RR was involved in regulation of gene expression of members of the proteolytic enzyme system (Azcarate-Peril et al., 2005). Studies of oligopeptide gene expression in lactobacilli are scarce. In L. delbrueckii ssp. bulgaricus no significant differences were detected in the amount of oligopeptide mRNA per total RNA in preparations from the early- and late-exponential phase cultures grown in whey broth culture (Peltoniemi et al., 2002). A recent study indicated that, in L. helveticus, growth in milk also significantly induced various peptidases, proteinases, and the oligopeptide transporters (Smeianov et al., 2007).

Sixteen peptidases showed noteworthy temporal patterns of expression in L. acidophilus. In contrast, only 9 peptidases were induced in L. helveticus (Smeianov et al., 2007) during growth in milk; however, in that study, gene expression was analyzed at one time point (9 h, pH 5.5) and not over the various growth phases. In L. acidophilus, only expression of oligoendopeptidase F (pepF, LBA1763) peaked at 4 h displaying a QC pattern of gene expression because most of the differentially expressed peptidases were mainly expressed at 12 h (early stationary phase). The pepF gene is well conserved among lactobacilli, and other members of the LAB group. The pepF gene encoded by LBA1763 shows a 49% identity with pepF from Lactococcus lactis. In this bacterium, PepF hydrolyzes peptides containing more than 7 and less than 17 amino acids (Monnet et al., 1994). These expression profiles suggest an early role of PepF breaking larger peptides into smaller ones that will be then processed by other peptidases of the proteolytic system. Additionally, several aa transporters showed increased levels of transcription over time (Table 2). Interestingly, the differentially expressed genes LBA1115 and LBA1902 coding for aa transporters appear to be unique to the acidophilus group (including L. acidophilus, L. gasseri, and L. johnsonii) and L. reuteri.

Most of the stress-related genes differentially expressed showed either a QV or a QLVU pattern, suggesting high levels of expression toward stationary phase during growth of L. acidophilus in milk. These trends are in accordance with the accumulation of lactic acid (and consequent decrease in pH) and other secondary metabolites. Several mutant strains were evaluated for their acidification activity, survival during storage conditions, and adhesive properties to mucin and Caco-2 cells. These derivatives contained mutations in genes found to be differentially expressed when L. acidophilus was grown in milk. As expected, the derivative with a mutation in lacL showed decreased acidification ability (as indicated by higher tm values) and decreased survival in acidified skim milk at 4°C, indicating that β-galactosidase activity is required not only for growth but also for survival under storage conditions. We observed increased tm values and reduced survival in acidified milk at 4°C for strains carrying mutations in LBA1524HPK, LBA0555 (annotated as a myosin-crossreactive antigen) and LBA1392, a mucus-binding protein precursor (mub) demonstrated to mediate adherence to intestinal epithelial cells (Buck et al., 2005).

Interestingly, luxS, responsible for the transformation of S-ribosylhomocysteine into homocysteine in the methyl activated cycle and the production of AI2 from methionine, showed similar trends of expression in milk as in MRS (Buck et al., 2008), with increased expression toward stationary phase. It is noteworthy that the luxS mutant strain showed increased tm values (although not significant at a P > 0.1 level) suggesting an important role for LuxS during growth in milk.

The adhesion capabilities of probiotic strains in the presence of milk have not been extensively analyzed. Ouwehand et al. (2001) reported reduced adhesion levels of L. brevis, L. reuteri, and L. rhamnosus upon exposure and growth in skim milk, in milk containing 1.5% fat, and in nonhomogenized milk with fat content of 1.6 to 1.9%. In our study, we observed comparable levels of adhesion when L. acidophilus NCFM cells were grown in skim milk or MRS. This is a technologically relevant fact because probiotic cultures are often consumed via milk-based food products. Additionally, milk, through its buffering action, is likely to provide a protective medium to probiotics that will increase the number of microorganisms surviving the passage through the stomach to reach the small intestine. Some genes putatively involved in adhesion were expressed differentially during growth of L. acidophilus in milk. Especially relevant is the overexpression of the genes encoding aggregation-promoting proteins, specifically the protein encoded by LBA0493, which is similar to the aggregation-promoting factor (APF) described in L. crispatus M247 (Marcotte et al., 2004). It has been shown that a nonaggregating isogenic spontaneous mutant derived from M247 was unable to colonize the intestinal tract suggesting a role of aggregation in colonization of the gastrointestinal tract (Cesena et al., 2001).

In the present study, the comparison of adhesion abilities of 6 mutants indicated that an unknown membrane protein (LBA1690) was involved in adhesion of L. acidophilus NCFM grown in milk to mucin. Detectable differences were also observed for the HPK mutant and LBA1690 to Caco-2 cells. Some histidine protein kinases from 2CRS have already been shown to be involved in regulation of adherence of gram-positive pathogens such as Streptococcus agalactiae to immobilized fibrinogen via the regulation of the expression of bacterial cell surface components (Spellerberg et al., 2002), and adhesion of L. plantarum WCFS1 to a glass surface via the overexpression of genes involved in cell surface polysaccharides synthesis (Sturme et al., 2005). The specific role of LBA1524HPK-LBA1525RR in adhesion might be related to the function of the 2CRS in the regulation of the transport of oligopeptides (Azcarate-Peril et al., 2005) as it was demonstrated in S. gordonii and S. parasanguis, where a mutation of the oligopeptide permease systems led to a decrease in bacterial cell adhesion [reviewed by Leonard and Podbielski, (1999)]. However, it is not clear whether the oligopeptide-binding proteins can act as adhesins themselves or if they are required for the transport of signaling peptides that impact adherence factors.

The data available on the hypothetical protein LBA1690 revealed the presence of a signal peptide and a transmembrane domain, suggesting that this protein is secreted and anchored to the membrane. Further studies are needed to determine the function of this protein and its potential role in adhesion. Surprisingly, the putative mucus-binding protein encoded by LBA1392, although differentially expressed during growth in milk, did not appear to have a role in adhesion during growth in milk, whereas this protein was previously shown to be involved in adherence on Caco-2 cells after the strain was grown in MRS (Buck et al., 2005). It might be speculated that growth in milk alters the cell surface display to promote attachment in spite of the absence of the LBA1392:mub protein.

Our study presents, for the first time, the analysis of the effect of milk, as a delivery vehicle, on the global gene expression of a probiotic strain, L. acidophilus. Additionally, genes involved with growth and survival in milk have been identified, some of which may be potentially important for probiotic traits.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study was supported by the Southeast Dairy Foods Research Center; Dairy Management, Inc.; and the North Carolina Dairy Foundation. We thank J. Bruno-Barcena (Department of Microbiology, North Carolina State University) and E. Durmaz (Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University) for scientific advice and helpful suggestions, and Jason Osborne, at the Department of Statistics (North Carolina State University), for his advice on microarray data analysis. We are grateful to the Genome Research Laboratory at North Carolina State University for technical assistance and staff that supported printing of the microarray slides.

	FOOTNOTES

 
¹ Current address: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

² Current address: SAF-ISIS, Aquitaine, France.

Received for publication June 15, 2008. Accepted for publication November 5, 2008.

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