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Applied and Environmental Microbiology, October 2005, p. 6260-6266, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6260-6266.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Proteolytic System of Lactobacillus sanfranciscensis DSM 20451T during Growth in Sourdough

Nicoline Vermeulen, Melanie Pavlovic, Matthias A. Ehrmann, Michael G. Gänzle,* and Rudi F. Vogel

Technische Universität München, Lehrstuhl Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany

Received 15 December 2004/ Accepted 29 April 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Wheat sourdough fermentation has a positive influence on the overall bread quality because it improves flavor (14, 29, 32) and texture (18) and prolongs shelf life due to the formation of antifungal compounds (20) and delayed staling (6). The fate of the protein fraction of the flour during sourdough fermentation is of crucial importance for bread quality. The protein network in wheat doughs determines dough rheology, gas retention, and thus bread volume and texture. Proteolytic events during fermentation provide the substrates for microbial growth and conversion of amino acids to flavor precursor compounds and antifungal metabolites (20, 32). Furthermore, some peptides from wheat proteins are involved in human cereal intolerance and their levels in bread hydrolysates are reduced by selected sourdough lactic acid bacteria (9).

The most abundant proteins in wheat flour are gluten proteins. After dough mixing, the proteins are linked to each other by disulfide and hydrogen bonds, resulting in an insoluble network with a molecular weight of up to several million (35). Proteolytic enzymes in the flour are associated with wheat gluten. The pH-optimum of activity of these aspartic proteinases is below 4.0 (2, 3). Accordingly, the proteolytic activity in wheat doughs is strongly increased in acidified doughs independent of the presence of lactic acid bacteria (32, 33) and proteolytic activity in sourdough extracts is inhibited by aspartic protease inhibitors but not by serine protease inhibitors (22). Thus, the most active proteases in wheat sourdoughs are the cereal aspartic proteinases whereas serine proteinases from lactobacilli play a minor role only (34). Preliminary evidence indicates that lactobacilli use peptides during growth in sourdough to meet their substrate requirements (9, 33).

The proteolytic system of lactic acid bacteria is best studied in Lactococcus lactis (for reviews, see references 5, 13, and 30). It employs an extracellular serine proteinase, Prt, which degrades casein to oligopeptides. Peptide uptake is preferred over amino acid uptake, and three different peptide transport systems were identified, Opp, DtpT, and Dpp. DtpT and Dpp are single proteins and transport hydrophilic di- and tripeptides. Opp is a system that consists of five proteins which mainly transports oligopeptides (8). Peptides are hydrolyzed by intracellular peptidases and further metabolized. This proteolytic system is required for the growth of L. lactis in milk because proteins are the only source of amino nitrogen and proteolytic activity in milk is low. In L. lactis, the expression of the proteinase and peptide transporters is reduced when an abundant supply of peptides or amino acids is present in the environment (13).

Although less extensively studied, the main features of the proteolytic system of lactobacilli appear to be similar to that one of L. lactis. The Prt proteinase has been characterized on genetic and biochemical level in several species of lactobacilli (30). The Opp transport system was genetically characterized in Lactobacillus delbrueckii (24); peptidases corresponding to enzymes from L. lactis are widely distributed among lactobacilli (5). From Lactobacillus sanfranciscensis, a dipeptidase and an aminopeptidase have been purified and characterized (12). Knowledge of the proteolytic system of lactobacilli is a prequisite for the optimization of bread quality through amino acid accumulation and microbial amino acid metabolism. However, the regulation of peptide transport enzymes and peptidases in sourdough lactobacilli has not been studied. We therefore aimed to identify genes in L. sanfranciscensis which are involved in proteolysis, peptide transport, and peptide hydrolysis to study their expression during growth in sourdough and to determine the effect of microbial metabolism on proteolysis and amino acid levels in dough.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strain and growth conditions.
Lactobacillus sanfranciscensis strain TMW1.53 (ATCC 27651T, DSM 20451T) was grown at 30°C in modified Man, Rogosa, and Sharp medium (mMRS; composition per liter, 5 g glucose · H2O, 10 g fructose, 10 g maltose, 10 g peptone from casein, 5 g meat extract, 5 g yeast extract, 4.0 g KH2PO4, 2.6 g K2HPO4 · 3H2O, 3.0 g NH4Cl, 1 ml Tween 80, 0.1 g MgSO4 · 7H2O, 0.05 g MnSO4 · H2O, 0.5 g L-Cys HCl · H2O, 0.2 mg each of biotin, folic acid, nicotinic acid, pyridoxal phosphate, thiamine, riboflavin, cobalamin, and panthothenic acid, 15 g agar for solid media). In N-limited mMRS, peptone and meat extract were omitted and 1 g liter–1 yeast extract was added as the sole source of complex nitrogen. The pH was adjusted to 6.2. Escherichia coli strain DH5{alpha} was grown aerobically at 37°C in Luria-Bertani broth containing ampicillin (100 mg liter–1) if necessary.

General molecular techniques.
Cloning, DNA manipulations, and agarose gel electrophoresis were done as described previously by Sambrook et al. (28). Chromosomal DNA was isolated from L. sanfranciscensis using an E.Z.N.A. bacterial DNA kit (Peqlab Biotechnologie GmbH, Erlangen, Germany). E. coli plasmid DNA was isolated with Montage Plasmid Miniprep 96 (Millipore, Schwalbach, Germany). Restriction endonucleases were obtained from Promega, (Mannheim, Germany) and MBI Fermentas (St. Leon-Rot, Germany), and T4 DNA ligase was obtained from Bioron GmbH (Ludwigshafen, Germany). PCRs were carried out with Taq polymerase from Qbiogene (Montréal, Quebec, Canada). PCR products were separated by gel electrophoresis, and bands of the expected size were isolated from the gel using an E.Z.N.A. gel extraction kit (Peqlab Biotechnologie GmbH) and sequenced by Sequiserve (Vaterstetten, Germany). Enzymes and reagents were used according to the manufacturer's recommendations unless otherwise stated.

Genome sampling of L. sanfranciscensis DSM 20451.
Chromosomal DNA of L. sanfranciscensis was digested with the restriction enzyme BsuRI or AluI or broken by ultrasonication for 40 s at 7 W on ice (Bandelin UW2070 ultrasonication processor). Fragments (800 to 1,500 bp) were isolated by gel electrophoresis. End repair of sonicated fragments was performed with a DNA terminator kit (Lucigen, Middleton, United Kingdom). DNA fragments were ligated into psmartHCAmp (Lucigen), cloned into E. coli strain DH5{alpha}, and sequenced by MWG Biotech (Ebersberg, Germany). Sequences were blasted in databases (WU-BLAST2 Search with postprocessing at EMBL [http://dove.embl-heidelberg.de/Blast2/] and translating BLAST at National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/BLAST/]). Sequence alignments and DNA-protein translations were performed using the online service of the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/ and http://www.ebi.ac.uk/emboss/transeq/, respectively). A total of 1,700 sequence runs resulted in contigs covering 20% of the genome.

Screening the L. sanfranciscensis genome for prt.
Degenerated primers were based on conserved prt sequences of L. lactis and lactobacilli (Table 1). The primers were targeted on six distinct highly conserved regions, taking into account the codon usage in the various prt genes of lactic acid bacteria.


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  		TABLE 1. Primers used for PCR detection of prt

 
Sourdough fermentations.
Wheat flour with an ash content of 0.55% was obtained at a local supermarket. To inoculate preferments, cells from 50 ml overnight culture were washed two times with tap water and resuspended in 5 ml tap water. Preferments were made of 25 g flour, 20 g tap water, and 5 ml cell suspension, stirred with a spatula, and incubated at 30°C for 16 h. Preferments were used to inoculate the sourdoughs which were prepared using the dough formulas shown in Table 2. Sourdoughs were incubated at 30°C, and samples were taken after 0 h, after fermentation to a dough pH of 4.8 (ca. 5 h), and after 24 h, corresponding to a pH of 3.6. In order to inhibit aspartic proteinases, pepstatin A (Sigma-Aldrich) was added to a final concentration of 40 µM from a stock solution in methanol-acetate (9:1) prepared daily.


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  		TABLE 2. Dough formulas for preparation of sourdoughs and chemically acidified doughs

 
Cell counts, amino nitrogen, and concentrations of metabolites in dough.
The concentrations of amino nitrogen as well as the metabolites lactate, acetate, and ethanol were determined using a modified ninhydrin method and high-performance liquid chromatography, respectively, as described by Thiele et al. (32). Dough samples were analyzed in duplicate, and the experimental error of the analytical methods was generally less than 10%. Cell counts were determined by plating on mMRS agar (32).

Determination of proteolytic activity of sourdoughs.
The proteolytic activity of sourdough systems was investigated by monitoring the hydrolysis of fluorescent casein prepared according to the method of Twining (34). The use of fluorescein isothiocyanate (FITC)-casein was preferred over the use of FITC-gliadin or -glutenin to avoid problems associated with gluten solubility in dough and laboratory media (33, 36). Dough formulas are shown in Table 2. The doughs were incubated at 30°C for 5 and 24 h. To 200 mg sourdough, 300 µl distilled water and 20 µl 70% perchloric acid were added in order to precipitate proteins. After overnight precipitation, solids were removed by centrifugation. Supernatants were neutralized by the addition of 4 volumes of Tris-HCl buffer (pH 8, 1 M). In order to determine the fluorescence-labeled hydrolysis products of fluorescent casein, the fluorescein fluorescence intensity was measured in a microtiter plate spectrophotometer (TECAN spectrafluor, Grödig, Austria).

Total RNA isolation and copy DNA (cDNA) synthesis.
One hundred grams of sourdough was suspended in 200 ml Tris-HCl buffer (50 mM, pH 7.0) and centrifuged to remove large particles (5 min, 1,500 relative centrifugal force). Cells of L. sanfranciscensis predominantly remained in the supernatant and were harvested by centrifugation (15 min, 4,500 relative centrifugal force) and resuspended in 3 ml Tris-HCl buffer (50 mM, pH 7.0) with 3 ml RNAprotect (QIAGEN, Hilden, Germany). This cell suspension was used for RNA isolation with the QIAGEN RNeasy Mini kit. DNA was removed by treating the sample with RQ1 RNase-free DNase (Promega). Reverse transcription was performed by incubation of RNA with random hexamer primers (random hexadeoxynucleotides) at 70°C for 10 min. After cooling on ice, 1 µl each of deoxynucleoside triphosphates (25 mM), 1 µl reverse transcriptase (200 U µl–1, Moloney murine leukemia virus-RT, RNase H minus; Promega), 5 µl reaction buffer (supplied with reverse transcriptase), and 5µl RNase-free water were added. The sample was incubated at 25°C for 10 min and subsequently at 42°C for 110 min, and the reaction was stopped by heating the sample at 72°C for 15 min.

Real-time PCR.
Real-time PCR was performed in a LightCycler instrument (Roche Molecular Biochemicals, Mannheim, Germany). The primers used for real-time PCR are shown in Table 3. The reaction mixture consisted of 10 µl QuantiTect SYBR green PCR Master Mix (QIAGEN), 2 µl cDNA, 6 µl DNase-free water, and 1 µl of each primer (final concentration, 0.5 pM). The PCR was carried out in glass capillaries. Crossing points (CP, the point at which the fluorescence rises appreciably above the background fluorescence [26]) were determined by using the Fit Point Method in Roche LightCycler software 3.5. In order to determine the real-time PCR efficiency, standard curves using diluted chromosomal DNA were included for each primer pair. The efficiency (E) was calculated from the slopes given by the LightCycler software according to the equation E = 10(–1/slope) (26). In cDNA libraries, the amount of the respective target genes was calculated relative to their amount in the control, i.e., the cDNA libraries prepared from cells exponentially growing in sourdough without additives. To account for variation in the mRNA isolation and cDNA synthesis, differences in the amounts of target genes were normalized against the differences in the ldh according to the following equation (26):

Etarget and Ereference represent the efficiencies of the respective PCRs, and target genes were oppF, dtpT, and pepT. ldhL, coding for the lactate dehydrogenase, was used as a reference gene in order to relate the transcription of the target genes to the central carbon metabolism. ldh was preferred as a reference over rRNAs to avoid the comparison of stable, high-copy-number rRNA with fast degradable, low-copy-number mRNA (7). The efficiencies of the PCRs were determined as 1.61, 1.78, 1.89, and 2.15 for oppF, dtpT, pepT, and ldh, respectively.


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  		TABLE 3. Oligonucleotide pairs used for determining gene expression of L. sanfranciscensis DSM 20451

 
Nucleotide sequence accession numbers.
Sequences were submitted at EMBL Nucleotide Sequence Submissions (http://www.ebi.ac.uk) and have the following accession numbers: for dtpT, AJ866920; oppF, AJ866923; oppD, AJ866924; oppA-pepN, AJ866928; pepX, AJ866927; pepR, AJ866925; pepT, AJ866926; pepC, AJ866922; and ldh, AJ866921.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening for proteolytic activity of L. sanfranciscensis on the biochemical and genetic level.
To determine whether L. sanfranciscensis strain DSM 20451 exhibits proteolytic activity that enables the strain to use proteins as the sole source of complex nitrogen, the strain was inoculated on N-limited mMRS or N-limited mMRS additionally containing 3 g liter–1 casein or 1.25 g liter–1 peptone from casein. Growth was observed on N-limited mMRS in the presence of peptides but not in the presence of casein only or in the absence of an additional amino acid source (data not shown). This indicated that L. sanfranciscensis strain DSM 20451 does not exhibit proteolytic activity strong enough to enable the strain to grow on proteins as the sole source of complex nitrogen. Because sequences corresponding to Prt proteinases carrying out protein hydrolysis in other lactic acid bacteria were not obtained on the genome preview, chromosomal DNA of L. sanfranciscensis DSM 20451 was specifically screened for the presence of a prt gene by using eight different primer combinations (Table 1). All primer combinations amplified prt fragments of appropriate sizes when DNA from Lactobacillus helveticus strain TMW1.1255 harboring prtH or L. lactis strain NCDO 712 harboring prtP was used as a template in the PCRs. Two of the PCR products were sequenced to ensure the suitability of the primer pairs used for the screening. PCR products of the appropriate sizes were not observed when DNA from L. sanfranciscensis was used as a template, and sequencing of unspecific PCR products did not reveal any similarity to prt genes or other proteinases.

Proteolytic activity of L. sanfranciscensis during growth in sourdough.
To determine a possible influence of L. sanfranciscensis strain DSM 20451 on proteolytic events during sourdough fermentations, the strain was inoculated in wheat doughs. The strain grew to high cell counts in all doughs, and the identity of the fermentation microflora with the inoculated strain was verified by the observation of a uniform colony and cell morphology (data not shown) and specific PCR (see below). Pepstatin A was used to inhibit the proteolytic activity of endogenous aspartic proteinases, and fermented doughs were compared to aseptic acidified doughs. Proteolysis was assessed by the determination of amino nitrogen levels and the degradation of fluorescent casein. The levels of the metabolites lactate, acetate, and ethanol (Table 4) indicated that pepstatin A had no appreciable inhibitory effect on the growth and metabolism of L. sanfranciscensis. The acetate in the pepstatin A stock solution enhanced acetate levels in dough. Cell counts in aseptic doughs were below 105 CFU g–1, excluding an impact of microbial metabolism on proteolytic events in these doughs. The amino nitrogen levels of fermented and aseptic doughs are shown in Fig. 1A. In aseptic acidified doughs, amino nitrogen levels roughly doubled during incubation due to the activity of endogenous aspartic proteinases. The levels of amino nitrogen were higher in sourdoughs than those in aseptic acidified doughs, but in both, the release of amino nitrogen during fermentation was virtually inhibited by the addition of pepstatin A. The largest increase of free amino nitrogen was observed in sourdoughs with the addition of peptides. In Fig. 1B, the changes in fluorescence during the fermentation of doughs with fluorescent casein are depicted. In the aseptic acidified dough, casein was degraded by endogenic flour proteinases, resulting in increased fluorescence in aqueous extracts of low-molecular-weight peptides and amino acids. In agreement with previous results obtained with fluorescent gliadin and glutenin (33), dough fermentation with L. sanfranciscensis slightly increased protein degradation compared to the aseptic acidified dough, but the addition of pepstatin A inhibited protein degradation to a large extent in both aseptic and microbially fermented doughs. The release of peptides from fluorescent casein in aseptic doughs and sourdoughs with pepstatin A did not correspond to a comparable increase of amino nitrogen (Fig. 1A and B). Large peptides have only one free amino nitrogen group detected by the ninhydrin staining but may have several fluorescent groups. Therefore, residual proteolytic activity remaining in the presence of pepstatin A appears to produce rather large peptides. Taken together, proteolysis in the sourdoughs cannot be attributed to a serine proteinase L. sanfranciscensis. Increased levels of amino nitrogen or amino acids in sourdoughs compared to aseptic doughs were observed only when cereal proteinases were active, i.e., in the absence of pepstatin A, and are thus attributable to the hydrolysis of peptides rather than proteins.


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  		TABLE 4. Metabolite concentrations in sourdoughs fermented for 24 h with L. sanfranciscensis DSM 20451a

 


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  		FIG. 1. Proteolytic activity in sourdoughs fermentated with L. sanfranciscensis strain DSM 20451. The following doughs were analyzed: C, chemically acidified doughs; C-A, chemically acidified doughs with aspartic protease inhibitor; SF, L. sanfranciscnesis sourdough; SF-A, L. sanfrancsicensis sourdough with aspartic protease inhibitor; and SF-Pe, sourdough with the addition of peptides. (A) Accumulation of low-molecular-weight amino nitrogen. Amino nitrogen was determined after 0, 5, and 24 h of fermentation with a modified ninhydrin method. Presented are average values ± the standard deviations of two separate fermented doughs. (B) Degradation of FITC-labeled casein. The fluorescence of trichloroacetic acid-soluble degradation products was determined as arbitrary fluorescence units (AU) after 0, 5, and 24 h of fermentation.

 
Identification of genes related to peptide transport and hydrolysis in L. sanfranciscensis strain DSM 20451.
Because L. sanfranciscensis strain ATCC 27653 requires peptides for growth (1), we considered enzymes involved in peptide transport and hydrolysis of crucial importance. The genome of L. sanfranciscensis was partially sequenced, and partial open reading frames were identified with high similarities to peptide transport systems and peptidases of lactobacilli and L. lactis (Table 5). Two peptide transporters, Opp and DtpT, and five peptidases, PepC, PepX, PepR, PepT, and PepN, were identified. In other lactobacilli, Opp is encoded by five genes (oppD, oppF, oppB, oppC, and oppA) with monocistronic organization (24). In L. sanfranciscensis, the gene encoding PepN is located 150 base pairs downstream from oppA and a PCR approach using forward and reverse primers located on the oppA and pepN genes using copy DNA as a template confirmed that these two genes are located on the same operon (data not shown).


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  		TABLE 5. Partial sequences from L. sanfranciscensis DSM 20451 coding for putative proteins involved in peptide transport or hydrolysis

 
Transcription of pepR, pepC, pepX, pepT, opp-pepN, and dtpT and regulation of opp-pepN, dtpT, and pepT by L. sanfranciscensis during sourdough fermentation.
In order to investigate whether the peptide transport systems were relevant for peptide metabolism by L. sanfranciscensis during growth in sourdough, we studied the expression of genes involved in peptide uptake and hydrolysis on transcriptional level. Transcripts of the genes dtpT, oppF, pepC, pepR, and pepT were detected in cDNA libraries from L. sanfranciscensis growing exponentially in sourdough, whereas transcripts of the gene pepX were not detected. To evaluate the role of the peptide supply on gene expression, doughs to which peptone from casein was added were analyzed in addition to doughs without additives. Samples for total RNA isolation were taken during the exponential (pH 4.8) and stationary (pH 3.6) growth phases. The genes coding for peptide uptake systems, oppF and dtpT, and additionally, one peptidase, pepT, were chosen for semiquantitative transcriptional analysis. The regulation of gene expression in sourdoughs was investigated by quantification of the respective transcripts relative to ldh in the four cDNA libraries by using real-time PCR (Table 6). The expression of peptide transporters was highest in cells growing exponentially in dough without externally added peptides, and the transcription of dtpT and of opp-pepN was reduced 31- and 71-fold, respectively, in the stationary phase. The dtpT and opp-pepN transcription by stationary cells in doughs with peptides equaled the transcription by stationary cells of L. sanfranciscensis in dough without peptides. However, the addition of peptides decreased the transcriptional levels of dtpT and opp-pepN in exponentially growing cells 16- and 18-fold, respectively.


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  		TABLE 6. Expression of genes related to peptide transport and hydrolysis by L. sanfranciscensis DSM 20451 during growth in sourdough

 
The effects of the growth phase and the peptide supply on pepT transcription are much smaller than the effects on dtpT and opp-pepN transcription (Table 6). In dough without additives, transcription was reduced 3.5-fold in stationary cells compared to exponentially growing cells. The addition of peptides decreased pepT expression in exponentially growing cells, but in the stationary growth phase, the transcriptional levels of pepT were higher when peptides were present.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteolytic activity of L. sanfranciscensis.
The results presented here failed to provide evidence for the presence of cell wall-associated protease in the L. sanfranciscensis type strain. First, L. sanfranciscensis was not able to grow on casein as the sole nitrogen source. Previously, a screening of 108 sourdough isolates has shown that only five exhibited proteolytic activity, indicating that proteolytic activity is strain dependent and that the lack of cell wall-associated proteinases is the rule rather than the exception in sourdough lactic acid bacteria (25). Second, proteolytic activity in aseptic doughs and sourdoughs was inhibited upon the addition of an aspartic protease inhibitor, indicating that cereal proteinases are responsible for the first step in gluten degradation. Aspartic protease inhibitors but not serine proteinase inhibitors were previously reported to inhibit proteolytic activities in sourdough extracts (22). Third, screening the genome of L. sanfranciscensis strain DSM 20451 did not provide evidence for a prt-like proteinase. Likewise, the Lactobacillus plantarum strain WCFS1 genome does not contain prt. A comparison of the genomes of Lactobacillus johnsonii strain LA-1 and L. plantarum indicated that their metabolic potential related to peptide uptake, peptide hydrolysis, and amino acid biosynthesis reflects the adaptation to nutrient-rich or -poor environments, respectively. The L. plantarum harbors no prt and fewer peptidase genes but retains more amino acid biosynthetic capability (4, 16). Biochemical and genetic studies in dairy lactobacilli such as Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis (15) as well as L. lactis (13) demonstrate that these organisms reduce the expression of proteinases when sufficient peptides are present in its environment. Milk is poor in amino acids and peptides and has low proteolytic activity. Therefore, proteinase activity (Prt) is a prerequisite for the growth of lactic acid bacteria in milk. In contrast, cereals contain higher levels of amino nitrogen and have a high proteolytic activity and thus support the growth of nonproteolytic lactobacilli.

Gobbetti et al. (12) purified and characterized a 57-kDa cell envelope serine proteinase from L. sanfranciscensis strain CB1 grown in a gluten-containing medium. Prt proteinases from lactic acid bacteria have a relative molecular weight of 135 to 145 (30), but proteinases with relative molecular weights of 45 and 180 are also reported in the literature (19). Poquet et al. (27) noticed that the prt-deficient L. lactis strain MG1363 has extracellular proteolytic activity that was attributed to HrtA, the sole cell surface housekeeping serine protease involved in protein maturation and turnover. Additionally, HtrA is a key factor to the response to several stress conditions in L. lactis (11) and in L. helveticus (31). HtrA from E. coli was characterized as a serine protease with a relative molecular weight of 48, which is, under physiological conditions, active as a homododecamer (17, 21, 23). However, the monomers displayed endopeptidolytic activity toward casein (21).

Peptide utilization and regulation of peptide transport and hydrolysis.
Analysis of peptide and amino acid levels in sourdough has shown that L. sanfranciscensis strain LTH 2581 uses peptides to meet its nitrogen demand during growth in sourdough (32, 33) and L. sanfranciscensis strain ATCC 27653 requires peptides for optimum growth (1). We observed that chemically defined peptides but not amino acids support the growth of L. sanfranciscensis strain LTH 2581 on N-limited mMRS (data not shown). This indicates that L. sanfranciscensis is the auxotroph for least one amino acid which must be transported into the cell as part of peptides. Genes encoding two peptide transport systems, dtpT and opp, and five peptidases, pepC, pepX, pepN, and PepT, were found on the genome of L. sanfranciscensis strain DSM 20451 (Fig. 2). It was shown that Opp, DtpT, PepT, PepC, PepR, and PepN are expressed during sourdough fermentation.



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  		FIG. 2. Schematic overview of the peptide utilization of L. sanfranciscensis DSM 20451 during sourdough fermentation retrieved by a genome-sampling approach. Opp, oligopeptide transport system; DtpT, di- and tripeptide transport system. Proteins printed in bold refer to genes transcribed during sourdough fermentation.

 
The addition of peptides to dough resulted in the reduced transcription of both dtpT and opp-pepN in exponentially growing cells. During the stationary growth phase, dtpT and opp-pepN transcription are reduced regardless of the addition of peptides. Thus, the high expression of peptide transporters in exponentially growing cells is attributable to the limited peptide supply in dough rather than increased nitrogen requirements during exponential growth. Because amino acids and peptides accumulate during fermentation, externally added peptides no longer affect the expression of peptide transporters during stationary phase. The regulation of the PepT peptidase upon the addition of peptides was much less pronounced. Peptide availability is therefore a limiting factor in amino acid metabolism.

The putative proteins identified in L. sanfranciscensis are highly similar to the corresponding proteins in L. lactis. In L. lactis, the proteolytic system consists of 3 peptide transporters and 17 intracellular peptidases (13). Because only 20% genome coverage was achieved with our approach, additional peptidases and/or peptide transport systems are likely to be present in L. sanfranciscensis. Our findings on the organization and regulation of the proteolytic system of L. sanfranciscensis only partially overlap with L. lactis (13). In both organisms, Opp expression was strongly reduced by the addition of peptides and PepT expression was reduced two- to fivefold. DtpT was regulated in response to the peptide supply in L. sanfranciscensis but not in L. lactis.

The levels of amino nitrogen in L. sanfranciscensis sourdoughs were higher than those in an aseptic acidified dough. This difference was more pronounced when peptides were added to the dough. Similar observations have been made by Corsetti et al. and Di Cagno et al. (6, 10), who reported that sourdough fermentation results in a more complete degradation of peptides than an aseptic acidifed dough. This implies that the difference between L. sanfranciscensis-fermented doughs and aseptic acidified doughs depends on a synergistic effect between flour proteinases and the peptide metabolism of L. sanfranciscensis. The peptides are taken up by the cells and hydrolyzed, and a part of the amino acids is used for further metabolism. Remaining amino acids which are not needed may be excreted into the dough.

In conclusion, we partially characterized the proteolytic system of L. sanfranciscensis strain DSM 20451 based on a genome-sampling approach and the determination of gene expression during growth in sourdough. Although the genes of L. sanfranciscensis which are involved in peptide transport and metabolism are highly similar to those of L. lactis, there are differences in the organization of these genes and in their regulation. L. sanfranciscensis DSM 20451 does not have a proteolytic activity which contributes to the hydrolysis of proteinaceous substrates. L. sanfranciscensis needs peptides for growth during sourdough fermentations which originate from protein hydrolysis by the endogeneous flour proteinases. Metabolism of peptides by L. sanfranciscensis may result in the accumulation of smaller peptides, amino acids, and amino acid metabolites which contribute directly or indirectly to the characteristics of sourdough bread.


   ACKNOWLEDGMENTS
 
We thank Claudia Thiele for providing fluorescent casein.


   FOOTNOTES
 
* Corresponding author. Present address: Food Microbiology and Probiotics, Department of Agricultural, Food and Nutritional Science, 4-10 Ag/For Centre, University of Alberta, Edmonton, AB T6G 2P5, Canada. Phone: (780) 492-0774. Fax: (780) 492-4265. E-mail: michael.gaenzle{at}ualberta.ca. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, October 2005, p. 6260-6266, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6260-6266.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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