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Applied and Environmental Microbiology, October 2005, p. 5873-5878, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5873-5878.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inducible Gene Expression in Lactobacillus reuteri LTH5531 during Type II Sourdough Fermentation

Fabio Dal Bello,¹ Jens Walter,² Stefan Roos,³ Hans Jonsson,³ and Christian Hertel^1*

Institute of Food Technology, University of Hohenheim, Stuttgart, Germany,¹ Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand,² Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden³

Received 16 December 2004/ Accepted 29 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactobacillus reuteri LTH5531 is a dominant member of the microbiota of type II sourdough fermentations. To investigate the genetic background of the ecological performance of LTH5531, in vivo expression technology was used to identify promoters that show elevated levels of expression during growth of this organism in a type II sourdough fermentation. Thirty-eight sourdough-induced fusions were detected, and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g., acid and general stress response), reflecting the harsh conditions prevailing during sourdough fermentation. Further, eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway is of technological interest, as they are potentially involved in the formation of aroma precursors. Our study allowed insight into the transcriptional response of Lactobacillus reuteri to the dough environment, which establishes the molecular basis to investigate bacterial properties that are likely to contribute to the ecological performance of the organism and influence the final outcome of the fermentation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sourdough is an intermediate product in bread production and contains a microbiota comprised of lactic acid bacteria and yeasts (reviewed in reference 16). Microbiological studies have revealed that 43 species of lactic acid bacteria, mostly species of the genus Lactobacillus, and more than 23 species of yeast occur in this ecological niche. The metabolic activity of these microorganisms leads to an acidification of the dough and the development of aroma precursors and is therefore of major importance for the quality of the final product. Sourdough breads are characterized by their unique flavor and texture, enhanced nutritional value, and favorable technological properties, such as prolonged shelf life and delayed staling (reviewed in reference 15). Type II sourdoughs serve mainly as dough acidifiers and are fermented for long periods (up to 5 days) at temperatures of >30°C and with high dough yields to permit pumping of the dough. Strains of Lactobacillus reuteri have been shown to be highly competitive in type II sourdough fermentations and persist over several years of continuous propagation in industrial fermentation processes (13, 22). Numerous ecological factors affect the competitiveness of lactobacilli in sourdough fermentations, i.e., temperature, ionic strength, dough yield, and microbial products, such as lactate, acetate, CO₂, and ethanol as well as factors resulting from substrates present in the cereal fraction and from enzymatic reactions (6, 22). The properties and genetic background responsible for the ecological performance of L. reuteri in sourdough fermentation are, however, poorly understood.

In vivo expression technology (IVET) has proved to be a valuable tool for dissecting bacterial adaptation to various environments and in the identification of colonization determinants (12, 25, 32). IVET permits the identification of promoters that are selectively induced in a particular habitat and has been used to identify genes of L. reuteri 100-23 that have elevated expression during colonization of the murine gut (42). It has been argued by Rainey (25) that genes showing greater expression in a particular ecosystem (niche-specific genes) are more likely to contribute towards ecological fitness than genes expressed equally across a range of environments. This assumption has been confirmed for a variety of in vivo-induced (ivi) genes (12, 25, 32). For example, the ivi gene for methionine sulfoxide reductase B (MsrB) was shown to contribute to the ecological performance of L. reuteri strain 100-23 in the gut of mice (43).

In this paper we describe the application of IVET to investigate in vivo gene expression of L. reuteri LTH5531 during type II sourdough fermentation. This strain has been isolated from the dominant Lactobacillus biota of a type II sourdough (22). Our results showed that 38 promoters are selectively expressed in LTH5531 during the fermentation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and media.
The Lactobacillus strains used in this study are listed in Table 1. Bacteria were routinely cultured in MRS medium (Difco) anaerobically or microaerobically (2% O₂, 10% CO₂, 88% N₂) at 37°C. When required, erythromycin and chloramphenicol were added to the culture medium at a concentration of 100 µg/ml and 10 µg/ml, respectively. Modified MRS (mMRS) medium contained, per liter, 10.0 g glucose, 5.0 g peptone, 4.0 g beef extract, 5.0 g Na-acetate, 2.0 g yeast extract, 2.0 g K₂HPO₄, 2.0 g triammonium citrate, 0.2 g MgSO₄, 0.05 g MnSO₄, 1 ml sorbitan monooleate, 0.5 g lichenan (Sigma), and 10 µg/ml chloramphenicol.

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TABLE 1. Bacterial strains and plasmids used in this study

Genetic techniques.
DNA manipulation methods were used according to standard protocols described by Sambrook et al. (29). Plasmid isolation, electrotransformation, and determinations of in vitro stability of plasmids pJW100 and pJW200 were performed as described previously (42).

Construction of IVET library.
A library containing DNA fragments (0.3 to 1.5 kbp) of the L. reuteri LTH5531 genome was constructed in the promoter trap vector pJW100 as described previously with some modifications (42). The high electrotransformation efficiency of LTH5531 allowed the direct establishment of the library in this strain, and the intermediate host (Escherichia coli) was omitted. The ligation reaction (pJW100 plus chromosomal DNA of LTH5531) was therefore used to electrotransform cells of LTH5531 directly. Transformants were grown microaerobically on mMRS agar at 37°C for 18 h. To determine in vitro promoter activity, Congo red solution (17) was introduced beneath the agar. ß-Glucanase activity (active promoter) was indicated by yellow zones (halos) surrounding the bacterial growth on an otherwise-red-colored plate (36). To reduce the number of strong constitutive promoters in the IVET library, transformants showing halos smaller than 2 mm in diameter were picked and cultivated on MRS agar containing chloramphenicol. These colonies were recovered from agar plates by using saline, and cell suspensions were subjected to plasmid isolation. The pooled plasmid preparation was used to generate the IVET library in L. reuteri LTH5531 by electrotransformation. To determine the average insert size, 20 colonies were picked randomly for plasmid isolation from MRS agar plates. An aliquot (1 µl) of the DNA solution was subjected to PCR (see below).

Inoculation and performance of sourdough fermentations.
L. reuteri LTH5531 containing the IVET library and the control strains FDB200, Con1, FDB100, and Pre1 (Table 1) were cultivated on MRS agar containing chloramphenicol (and erythromycin for FDB200 and Con1) for 48 h. Cells were recovered from the agar plates using 3 ml saline, and the cell suspensions were adjusted to an optical density at 600 nm of 20. An aliquot (100 µl) of the suspension (about 5 x 10⁸ cells) was used to start separated fermentations. Type II sourdough fermentations were performed as described by Meroth et al. (22), but lincomycin was added at a concentration of 0.5 g/kg dough. Briefly, batches of dough (200 g) were prepared from tap water and rye bran, providing a dough yield of 367 [(mass of dough/mass of flour) x 100]. Fermentation was started by the addition of 100 µl of inoculum (about 5 x 10⁸ L. reuteri cells) followed by incubation at 40°C for 24 h, stirring at 200 rpm. Thereafter, the dough was propagated by back-slopping of 1% of ripe dough and incubation for a further 24 h. A sample of 1 g was subjected to microbial counting on agar plates supplemented with 0.1 g/liter cycloheximide and the appropriate antibiotics. From the control batches, lactobacilli were recovered on MRS agar containing chloramphenicol (and erythromycin for FDB200 and Con1) with incubation at 37°C for 48 h, microaerobically.

Detection of ivi genes during sourdough fermentation.
From the batch inoculated with LTH5531 containing the IVET library, lactobacilli were grown on mMRS agar incubated for 18 h at 37°C, microaerobically. Approximately 3,000 colonies were screened for ß-glucanase activity (in vitro-active promoter) by observing the halo size on the mMRS agar plates. Clones with reduced or no halos were subcultured and stored at –80°C. The erythromycin resistance of these ß-glucanase-negative clones was determined by comparing growth on MRS agar supplemented with chloramphenicol (10 µg/ml) and erythromycin (100 µg/ml) to that of the control culture, L. reuteri FDB200. Finally, to confirm in vivo induction of genes, putative ivi clones were used to inoculate small batches of sourdough (5 g) containing 0.5 g of lincomycin/kg.

Analysis of ivi fusions.
Plasmid inserts of putative ivi clones were amplified by PCR using primers IVETrrnT1T2 and IVETrev as described previously (42). PCR products were purified using the QIAquick PCR purification kit (QIAGEN) and sequenced with the IRD 800-labeled primer IVETrrnT1T2-800 and IVETrev-800 using the AutoRead sequencing kit (Amersham Pharmacia Biotech). Homology searches were performed against the NCBI database using the BLASTX program (http://www.ncbi.nlm.nih.gov/BLAST). Homology searches against the genome sequence of L. reuteri ATCC 55730 (which is estimated to cover 90 to 95% of the complete genome [unpublished data]) were performed using a local version of the BLASTN program.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of L. reuteri LTH5531 genes selectively expressed during sourdough fermentation.
The IVET system consists of the promoter-trapping vector pJW100 (Table 1), in which genomic DNA fragments of LTH5531 were inserted upstream of two promoterless reporter genes (42). The primary reporter gene (essential growth factor) was 'ermGT, which confers lincomycin and erythromycin resistance. Selection of active promoters was achieved by addition of lincomycin to the sourdough. The second reporter gene, 'bglM (encoding a ß-glucanase), allowed the differentiation between constitutive and ivi promoters. In vitro-active promoters could be sorted out by screening for ß-glucanase activity on mMRS agar plates. This system was tested with sourdough containing lincomycin by comparing an L. reuteri culture containing a constitutively expressed promoter cloned in pJW100 (FDB200) with that of a culture without a cloned promoter (FDB100). As shown in Fig. 1, FDB200 could grow in sourdough supplemented with lincomycin, whereas FDB100 could not be detected, indicating that pJW100 was suitable for use as a promoter trap vector to identify promoters that are active during sourdough fermentation.

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FIG. 1. Lactobacillus population in type II sourdoughs after 48 h of fermentation in the presence of lincomycin. Batches were inoculated with strains FDB200 and Con1 (positive controls), FDB100 and Pre1 (negative controls), or the IVET library (LTH5531 containing the IVET library). For strain descriptions, see Table 1.

Construction and screening of the IVET library.
Transformation of L. reuteri LTH5531 with DNA of pJW100 and pJW200 revealed efficiencies of about 10⁶ transformants per µg of plasmid DNA. This high transformability enabled the construction of the IVET library in L. reuteri LTH5531. Analysis of 20,000 transformants revealed that 9% exhibited a detectable ß-glucanase activity on mMRS agar plates, indicating the presence of a cloned in vitro-active promoter. As clones containing strongly expressed constitutive promoters might overgrow ivi promoters in the ecosystem, clones exhibiting a halo size of >2 mm on mMRS agar were discarded. One clone exhibiting a large halo (>4 mm; Con1) and one with a small halo (<2 mm; Pre1) were retained as control cultures (Table 1). A plasmid pool was derived from the remaining 18,600 clones, which subsequently constituted the IVET library in L. reuteri LTH5531. PCR analysis of plasmid DNA of 20 randomly picked clones revealed an average insert size of about 700 bp (range, 0.2 to 1.2 kbp).

Detection of selectively induced genes during sourdough fermentation.
To identify L. reuteri LTH5531 promoters induced during sourdough fermentation, a batch of dough was inoculated with 30,000 LTH5531 transformants. After 48 h of fermentation, the Lactobacillus population was comparable in size to that of batches inoculated with cultures of FDB200 and Con1 (Fig. 1), whereas negative control cultures of FDB100 and Pre1 did not grow in the sourdough. Screening of 3,000 clones for in vitro ß-glucanase activity detected 180 that were erythromycin sensitive and had reduced or no ß-glucanase activity. To confirm that they contained ivi promoters, each clone was tested for growth during sourdough fermentation in the presence of lincomycin. A total of 173 of these clones grew in the sourdough and achieved populations of about 2 x 10⁹ CFU/g dough.

Characterization of sourdough ivi promoters and genes.
Sequence analysis of the plasmid inserts in the 173 ivi clones revealed putative Lactobacillus promoters (–35 region, TTGACA; –10 region, TATAAT [21]; highly conserved bases are shown in bold) in the correct orientation to induce expression of the two reporter genes. Thirty-eight different promoters were detected, most on more than one occasion. In 29 of these sequences a ribosomal binding site and an open reading frame (ORF) were located downstream of the promoter. Fourteen ORFs could be annotated by alignment of sequences with those in public databases, and the results were confirmed by considering the corresponding sequences of complete ORFs available from the genome of L. reuteri ATCC 55730. Further, 13 ivi genes could be identified by comparison of the ORFs with the genome sequence of ATCC 55730. Finally, two ORFs were annotated by homology search in public databases but could not be found in the genome of ATCC 55730. The 29 ivi genes were grouped according to the standard Clusters of Orthologous Groups classification (35) and are listed in Table 2. The genes were involved in amino acid transport and metabolism (four ORFs), translation (three ORFs), nucleotide transport and metabolism (four ORFs), cell envelope biogenesis and outer membrane (three ORFs), intracellular trafficking and secretion (one ORF), energy production and conversion (one ORF), inorganic ion transport and metabolism (two ORFs), transcription (two ORFs), general functions (four ORFs), and unknown functions (five ORFs). In the case of the remaining nine sequences, neither any ORF downstream of the promoter nor any match with the genome sequence of L. reuteri ATCC 55730 was found.

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TABLE 2. L. reuteri genes that were induced during sourdough fermentation

Top Abstract Introduction Materials and Methods Results Discussion References

 
Application of the IVET library constructed from the genome of L. reuteri LTH5531 allowed the detection of promoters that are specifically induced during type II sourdough fermentation. Sequence analysis of the downstream-located ORFs revealed 29 ivi genes (Table 2) which are likely to influence the ecological performance of LTH5531 in sourdough. Two of these genes (ivi64 and ivi121) as well as nine insert sequences missing any ORF were not present in the genome of L. reuteri ATCC 55730, which is a human isolate. As the available sequence is estimated to cover 90 to 95% of the complete genome, the ivi genes or sequences may be located in the unsequenced regions. On the other hand, they may not be present in the genome of the human isolate and therefore are of special interest, since they could regulate expression of unique proteins of the sourdough isolate LTH5531 which are of ecological importance in the sourdough fermentation.

As type II sourdoughs are used as dough acidifiers, the fermentation aims to accumulate high amounts of lactic and acetic acid, accounting for a harsh environment requiring high acid tolerance of the bacteria. In the type of fermentation used in this study the final pH drops below 3.8 and the total titratable acids reach values of up to 80 (22). Strain LTH5531 responded to the acid stress by expressing the arginine deiminase (ADI) pathway as indicated by the detection of the ivi gene arcD (Table 2, ivi40). This is consistent with the observation that Lactobacillus sanfranciscensis, which is highly competitive in type I sourdoughs, also induces the ADI pathway during fermentation (8). Moreover, Rollan et al. (27) recently demonstrated that arginine metabolism in the sourdough isolate L. reuteri CRL 1098 contributes to the survival under acid conditions and that the ADI enzymes are triggered by adaptation to low pH and/or energy depletion in the stationary phase of growth. Degradation of arginine through this pathway leads to formation of ammonia, which increases the acid tolerance of the organism by neutralization of the environment. Furthermore, the guaA gene encoding a GMP synthase was found to be induced in strain LTH5531. Rallu et al. (26) showed that alteration in the guanine nucleotide pool is responsible for increased heat and acidic stress resistance in Lactococcus lactis.

Identification of other ivi genes associated with the bacterial stress response illustrates further that type II sourdough produced with rye bran constitutes a harsh habitat for L. reuteri. The ADP ribose pyrophosphatase belongs to the hydrolases of the nudix family (3). Genes encoding the nudix hydrolases are considered "housecleaning," because their function is to cleanse the cell of potentially deleterious endogenous metabolites and to modulate the accumulation of intermediates in biochemical pathways during the cell cycle or during periods of stress (3). Up to 30 nudix hydrolase genes are represented in the genome of Bacillus species (44), indicating their potential importance in cell function. Furthermore, ADP ribose pyrophosphatase has been shown to be a tellurite resistance factor in Rhodobacter sphaeroides (9). However, no data are available about contamination of rye bran by this metal. Gene ivi149 encoded a protein with 48% identity to the Lactobacillus johnsonii NCC 533 homologue of von Ebner's gland protein (Veg) (30), a member of the lipocalin family. Lipocalins are a diverse, poorly understood family of proteins composed, in the main, of extracellular ligand-binding proteins displaying high specificity for small hydrophobic molecules (5). In gram-negative bacteria, such as Escherichia coli, these proteins are anchored to the outer membrane, where they are thought to serve a starvation response function (4). However, sequence analysis of ivi149 did not reveal the presence of a signal peptide, which would have indicated an extracellular localization of the protein. Two ivi genes (uppS and murA1) were detected which are involved in the biosynthesis of peptidoglycan, a major component of the cell wall of gram-positive bacteria. An increased expression of these genes may be the response of strain LTH5531 to the harsh conditions of the sourdough environment. Recently, Piuri et al. (24) showed that modifications occur in the cell wall of Lactobacillus casei during osmotic stress. However, no data are available about a connection between cell wall modifications and acid stress in sourdough lactobacilli. To date, for lactobacilli only changes in the membrane composition have been observed (2, 31) and, in general, changes in the lipid profile of cell membranes have been reported to play a key role in the response of bacteria to environmental stresses (1, 11, 34).

The induction of some ivi genes permits us to draw conclusions on the availability of nitrogen sources in rye bran sourdoughs. The gene azlC encodes a transporter for branched chain amino acids, indicating the availability of branched chain amino acids during type II sourdough fermentation. This is consistent with the finding that free amino acids can accumulate during rye dough fermentation (18, 19, 37). However, L. reuteri LTH5531 also induced the gene asnB, which is involved in the synthesis of asparagine, thus indicating a deficiency of this amino acid in type II sourdough. Such a deficiency may be explained by the preference of sourdough lactobacilli to acquire amino acids via peptide transport (14, 20, 37), which may result in a bias of uptake of particular amino acids. To make use of the acquired peptides, a highly competitive sourdough strain should possess an active peptide hydrolase system. This assumption is consistent with the identification of ivi38, which encodes a metalloproteinase. Recently, Rollán and Font de Valdez (28) showed that the sourdough isolate L. reuteri CRL 1098 possesses an active peptide hydrolase system consisting of several metalloenzymes. Thus, the metalloproteinase that we have detected may be part of a peptide hydrolase system in LTH5531. Such a system could enable LTH5531 to make use of peptides as a nitrogen source. Proteolysis during sourdough fermentation would also be of importance in the generation of amino acids that are of relevance for flavor development in the baked goods (38). A further example of an aroma-relevant ivi fusion is arcD, which belongs to the ADI pathway. This pathway leads to the formation of ornithine, a precursor of the "roasty" aroma compound, 2-acetyl-pyrroline, formed during baking (33). We have observed (unpublished results) that degradation of arginine by Lactobacillus pontis by the ADI pathway led to accumulation of ornithine in the dough and, upon baking, of 2-acetyl-pyrroline in the crust, thus improving the sensory quality of wheat breads.

Two transcription regulators were found to be induced during sourdough fermentation. One of these belongs to the LytR family (ivi5), whose members are involved in regulation of toxin, bacteriocin, and exopolysaccharide (EPS) production (23, 45). Sourdough isolates of L. reuteri, including strain LTH5531 (unpublished results), were recently shown to produce EPS during sourdough fermentation (39). Up to now, no results have been published investigating the regulation of L. reuteri genes involved in EPS production. However, EPS production has been shown to be affected by environmental factors, like temperature and pH, e.g., in Lactobacillus helveticus ATCC 15807 (40, 41) and Streptococcus thermophilus 1275 (46). Therefore, it is tempting to speculate that L. reuteri LTH5531 may synthesize EPS in sourdough under control of the transcriptional regulator that we have detected. In situ EPS production is of technological relevance, since it affects dough rheology and bread texture (39). Moreover, EPS produced by sourdough lactobacilli, such as L. sanfranciscensis, has shown to exert a prebiotic effect by selectively supporting the growth of bifidobacteria (7).

Lactobacilli have been used for centuries in food preservation and are used with increasing intensity for specific industrial food fermentation processes. The results of this study show that IVET can be used to gain insight into the transcriptional response of lactobacilli during a food fermentation process. ivi genes were detected that have not previously been functionally characterized (e.g., the Veg protein) but might be essential for the ecological performance of lactobacilli in the food environment. In addition, genes (arcD, ivi38, and ivi5) were identified with the potential to influence the quality of the final bread in relation to aroma and nutritional value. Knowledge of the complex regulatory processes that occur in starter organisms during food fermentations will provide a molecular basis on which improved starter strains can be developed for industrial exploitation.

 
We thank M. Kranz and E. Focken for excellent technical assistance. We are indebted to Gerald W. Tannock for critical reading of the manuscript.

This work was financed by the Deutsche Forschungsgemeinschaft.

 
* Corresponding author. Mailing address: Institute of Food Technology, University of Hohenheim, Garbenstr. 28, D-70599 Stuttgart, Germany. Phone: 49 711 459 4255. Fax: 49 711 459 4199. E-mail: hertel{at}uni-hohenheim.de.

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Applied and Environmental Microbiology, October 2005, p. 5873-5878, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5873-5878.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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