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Appl Environ Microbiol, March 1998, p. 1059-1065, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Utilization of Oligopeptides by Listeria monocytogenes Scott A

Annette Verheul,dagger Frank M. Rombouts, and Tjakko Abee*

Department of Food Science, Food Chemistry/Microbiology Section, Agricultural University Wageningen, Wageningen, The Netherlands

Received 19 June 1997/Accepted 10 December 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226-233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The occurrence of Listeria monocytogenes in low numbers in raw and minimally processed foods may be unavoidable because of the pathogen's ubiquity and resistance properties. Food products that have been implicated in outbreaks of listeriosis mainly involve raw vegetables, meat products, and milk products, which contain low concentrations of free amino acids (11, 19, 27, 43, 48). Therefore, the multiple-amino-acid auxotroph L. monocytogenes must utilize alternative nitrogen sources for growth to high cell densities in those food products.

One of the virulence factors of L. monocytogenes is an extracellular metalloprotease (Mpl) that is responsible for the cleavage of a lecithinase proenzyme to its active form, which has a function in cell-to-cell spread (40, 42). Protein degradation specifically associated with this protease is undetectable in vivo, excluding a possible role of Mpl in food environments (10, 29, 32). Consequently, L. monocytogenes is probably dependent on other proteolytic systems that allow degradation of food proteins; examples include indigenous proteinases or proteinases from other microorganisms (6, 13, 26, 27, 48). Hydrolysis of food proteins results in a large number of different peptides, depending on the proteolytic system(s) present. Evaluation of the ability of L. monocytogenes to use peptides of different sizes to fulfil its essential-amino-acid requirements might give insight into the growth characteristics of this important pathogen in certain food products.

Di- and tripeptides have been shown to be nutritionally valuable in providing L. monocytogenes with essential amino acids. Translocation of these peptides occurs prior to hydrolysis by internally located peptidases, and evidence that a proton motive force (PMF)-driven carrier is responsible for the transport of di- and tripeptides in L. monocytogenes has been found (51). Information about the utilization of oligopeptides (peptides containing four or more amino acid residues) by the pathogen as a source of essential amino acids is presently lacking.

In this work, the mechanism of oligopeptide utilization was studied in detail. Results indicate that L. monocytogenes can use oligopeptides containing up to eight residues as a source of amino acids. In addition, the results point out that the pathogen possesses distinct systems for the transport of di- and tripeptides on the one hand and oligopeptides on the other. The significance of the results for the understanding of (enhanced) growth of L. monocytogenes in certain foods elicited by the presence of proteolytic bacteria such as Pseudomonas spp. and Bacillus spp., or lactic acid bacteria in fermented foods, is discussed.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and growth media. L. monocytogenes Scott A, Bacillus cereus VC2, and Pseudomonas fragi DSM 3456 were grown in brain heart infusion (BHI) broth or in a defined minimal medium (DM) as described elsewhere (41). The amino acids in DM (L-leucine, L-isoleucine, L-valine, L-methionine, L-arginine, L-cysteine, and L-glutamine) were replaced by Na-caseinate or beta -casein (0.9% [wt/vol]) as required. For the growth experiments with valine-containing peptides, valine was omitted from DM.

Growth measurements. Growth experiments with valine-containing peptides were performed at 30°C in microtiter plates. Peptides were used at a concentration of 0.1 mM, and cultures were inoculated with 104 to 105 L. monocytogenes cells per ml. Changes in absorption (optical density at 620 nm) (OD620) were measured in a kinetic microtiter reader (Reader 340 ATTC, SLT-Instruments, Salzburg, Austria). To prevent evaporation, the incubation mixtures (200 µl each) were covered with 50 µl of paraffin oil (Wacker Chemie, GmbH).

The interaction between L. monocytogenes and other bacteria was studied by different approaches. Firstly, the growth of L. monocytogenes in DM without amino acids containing hydrolyzed Na-caseinate or beta -casein (0.9% [wt/vol]) was measured. Hydrolyzed casein was obtained after incubation of 4.5% (wt/vol) Na-caseinate or beta -casein with protease from Bacillus licheniformis (Solvay protease L660) at a final concentration of 0.2% (vol/vol) for 20 min at 30°C in 50 mM potassium phosphate (pH 7.0) containing 5 mM MgSO4. Enzyme activity was stopped by boiling the reaction mixture for 5 min and then immediately cooling it on ice. Growth was recorded as OD620 at 30°C in microtiter plates as described above. In the second approach, B. cereus or P. fragi was grown in DM without amino acids containing 0.9% (wt/vol) Na-caseinate with agitation (150 rpm) in a shaker-incubator (Gallenkamp, Griffin Europe, Breda, The Netherlands) at 30 or 20°C, respectively. After 24 h, cells were removed by centrifugation and the supernatant was adjusted to pH 7 and supplemented with glucose, ferric citrate, and vitamins at the concentrations present in DM. Subsequently, this solution was filter sterilized and inoculated with about 105 to 106 cells of L. monocytogenes Scott A per ml and incubated at 30°C with shaking (150 rpm). Cell growth of L. monocytogenes was monitored by plate counting on tryptic soy agar (TSA). Finally, the growth of L. monocytogenes in DM with Na-caseinate (0.9% [wt/vol]) present as the sole source of nitrogen was monitored in the presence of B. cereus or P. fragi at 20°C with shaking. Standard selective growth media were used for the enumeration of L. monocytogenes, B. cereus, and P. fragi in these mixed cultures, all obtained from Oxoid (Basingstoke, Hampshire, United Kingdom). Numbers of L. monocytogenes cells were determined by using PALCAM agar base (Oxoid CM877) combined with PALCAM selective supplement (Oxoid SR150). Pseudomonas agar base (Oxoid CM559) combined with Pseudomonas CFC selective agar supplement (Oxoid SR103) was applied for the selection of Pseudomonas spp., and numbers of Bacillus cereus cells were determined by using B. cereus selective agar base (Oxoid CM617) combined with B. cereus selective supplement (Oxoid SR99).

Detection of protease activity. (i) Skim milk well assay. B. cereus and P. fragi were grown overnight in BHI broth at 20 and 30°C, respectively, and diluted 108 times. Subsequently, 30 µl of the diluted cell suspension was dispensed in a well of a skim milk agar (Oxoid) plate; wells were made by removing the agar with a 6-mm-diameter glass tube. Clearing zones, indicating casein hydrolysis, were measured after incubation at 30°C (B. cereus) and 20°C (P. fragi) for 48 h.

(ii) Release of TCA-soluble peptides from casein. B. cereus and P. fragi were grown overnight in DM at 20 and 30°C, respectively, and the cells were separated from the medium by centrifugation. The supernatant was filter sterilized with a 0.2-µm-pore-size filter (Schleicher and Schuell GmbH, Dassell, Germany), and 200 µl of the filtrate was mixed with 800 µl of 50 mM Tris-HCl (pH 7.5) buffer containing 0.8% sodium caseinate, 5 mM CaCl2, and 0.08% sodium azide. Cell pellet (originating from 1 ml of culture) was resuspended in 1 ml of the same buffer. Following incubation for 2 h at 30°C, 1 ml of a solution containing 0.1 M trichloroacetic acid (TCA), 0.22 M sodium acetate, and 1.886% (vol/vol) acetic acid was added and the sample was kept at room temperature for 30 min. Nonsoluble material was removed by centrifugation, and the relative concentration of the TCA-soluble peptides was assessed by measuring the absorbance of the supernatant at 275 nm against an appropriate blank.

Preparation of CE and concentrated supernatant. L. monocytogenes was grown in 200 ml of DM and harvested during logarithmic growth at an OD620 of 0.6. The supernatant was concentrated 200-fold by ultrafiltration (on ice) with an Amicon filter (cutoff, 10 kDa; Amicon Corp., Lexington, Mass.). Cells were washed twice in 50 mM potassium phosphate (pH 6.9) and resuspended to a final OD620 of about 20. Lysis was achieved by incubating the cell suspension with mutanolysin (65 U/ml) at 37°C for 30 min followed by 15 cycles of sonication on ice (one cycle consisted of 15 s of sonication and 45 s of resting) with a Sanyo Soniprep 150 (Gallenkamp, Leicester, United Kingdom). Cell extract (CE) was obtained after removal of cell debris by centrifugation of the disrupted cells (75,000 × g for 10 min at 4°C) with a Biofuge Fresco Eppendorf centrifuge (Heraeus Instruments, Ostenrode, Germany). Concentrated supernatant and CE were stored at -20°C until further use.

Analysis of peptidase activities in L. monocytogenes. (i) HPLC. Concentrated supernatant and CE from L. monocytogenes cultures were incubated at 37°C in potassium phosphate (pH 6.9)-5 mM MgSO4 with 0.5 mM peptide. At various time intervals, samples were taken and peptides and amino acids were analyzed by reversed-phase high-performance liquid chromatography (HPLC) after derivatization with dansyl chloride as described elsewhere (51).

(ii) Chromogenic substrates. Concentrated supernatant and CE from L. monocytogenes cultures were incubated with alanyl-prolyl-p-nitroanilide, glycyl-prolyl-p-nitroanilide, acyl-alanyl-alanyl-alanyl-p-nitroanilide, succinyl-alanyl-alanyl-prolyl-phenyl-alanyl-p-nitroanilide, and isoleucyl-prolyl-arginyl-p-nitroanilide. Peptidase assays were performed essentially as described elsewhere (31) in a microtiter well plate in 50 mM Tris-HCl buffer (pH 7.5) at 37°C with the substrates present at 1 or 2 mM, and the release of nitroanilide was recorded at 405 nm over a 2-h period.

Oligopeptide transport. L. monocytogenes Scott A was grown in BHI and harvested during mid-exponential growth. Subsequently, cells were washed with 50 mM potassium phosphate (pH 6 or 6.9, as required) with 5 mM MgSO4, concentrated to an OD620 of approximately 20, and stored on ice until use. For transport assays, cells (OD620, 2) were preincubated at 30°C for 5 min in the presence of 20 mM glucose, after which 0.3 mM peptide was added. Transport was monitored by determining extracellular concentrations of residual peptide after removal of the cells by centrifugation (75,000 × g for 30 s at 4°C) with a Biofuge Fresco Eppendorfcentrifuge (Heraeus Instruments) at various time intervals. Peptides and amino acids were dansylated and analyzed by reversed-phase HPLC as described elsewhere (51). In experiments in which the phosphate analog vanadate was used, the potassium phosphate was replaced by 50 mM HEPES, pH 7.5.

Measurement of the membrane potential and intracellular ATP concentration. The transmembrane electrical potential (Delta psi ) was determined with an electrode specific for the lipophilic cation tetraphenylphosphonium (final concentration, 4 µM), as described previously (44). Cells of L. monocytogenes were prepared for measurements as described above and incubated (OD620, 2) at 30°C in 50 mM potassium phosphate (pH 6.9) in the presence of 20 mM glucose. The intracellular ATP concentration was determined as follows. Cells were lysed by using dimethyl sulfoxide, and ATP concentrations were determined by using the Lumac (Landgraaf, The Netherlands) luciferase bioluminescence assay. The amount of omitted light was recorded with a Lumac biocounter M2500. In experiments in which the phosphate analog vanadate was used, the potassium phosphate was replaced by 50 mM HEPES (pH 7.5).

Protein determination. Protein concentrations were determined by the method of Lowry et al. (28).

Chemicals. Peptides, beta -casein, and Na-caseinate, containing alpha , beta , and kappa  casein, were obtained from Sigma Chemical Co., St. Louis, Mo., or Bachem Feinchemikalien AG, Bubendorf, Switzerland. Chromogenic substrates were purchased from Bachem or from Chromogenix AB, Mölndal, Sweden. All other chemicals were reagent grade and were obtained from commercial sources.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Growth on valine-containing peptides. Growth of L. monocytogenes Scott A on specific peptides was measured by using DM containing all essential amino acids except for valine, which was supplied in the form of a peptide. Several peptides containing between 2 and 10 amino acid residues, with valine at different positions in the peptide chain, were used. L. monocytogenes failed to grow in DM lacking valine (Fig. 1A), while addition of certain valine-containing di-, tri-, tetra-, penta-, hexa-, hepta-, and octapeptides to DM without valine resulted in restoration of growth (Fig. 1B to H). The valine-containing nona- and decapeptides used in our study were ineffective in stimulating the growth of L. monocytogenes in DM without valine (Fig. 1I and J). These results indicate that oligopeptides can function as a source of essential amino acids for L. monocytogenes and suggest a size restriction for peptide utilization of 8 amino acids.


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  		FIG. 1.   Valine-containing peptides as sources of amino acids essential for growth of L. monocytogenes. L. monocytogenes was cultured at 30°C, and growth was monitored spectrophotometrically by measuring OD620. (A) Growth in DM (bullet ) and in DM without valine (open circle ). (B) Growth in DM without valine with dipeptides Val-Gly (black-triangle) and Ala-Val (triangle ). (C) Growth in DM without valine with tripeptides Val-Pro-Leu (black-square) and Ala-Val-Leu (square ). (D) Growth in DM without valine with tetrapeptides Val-Gly-Asp-Glu (black-lozenge ) and Val-Ala-Ala-Phe (diamond ). (E) Growth in DM without valine with the pentapeptide Val-Leu-Ser-Glu-Gly (black-down-triangle ). (F) Growth in DM without valine with hexapeptides Val-Gly-Gly-Ser-Glu-Ile (bullet ) and Gly-Ala-Val-Ser-Thr-Ala (open circle ). (G) Growth in DM without valine with the heptapeptide Arg-Val-Tyr-Ile-His-Pro-Phe (black-triangle). (H) Growth in DM without valine with octapeptides Val-His-Leu-Thr-Pro-Val-Glu-Lys (black-square) and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (square ). (I) Growth in DM without valine with nonapeptides Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (black-lozenge ) and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr (diamond ). (J) Growth in DM without valine with the decapeptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (black-down-triangle ). n, number of amino acid residues in peptides.

Peptidase activity in CE of L. monocytogenes. If oligopeptide-hydrolyzing enzymes are present externally, amino acids and di- and tripeptides which can subsequently be taken up by the corresponding transport systems will be formed (51), whereas intracellular accumulation of oligopeptides implicates the presence of a functional oligopeptide transport system. Incubation of CE obtained from DM-grown L. monocytogenes with hexa-alanine [(Ala)6] resulted in the appearance of alanine in the assay mixture (Fig. 2A). This conversion of (Ala)6 into alanine corresponds to a peptidase activity of 2 nmol min-1 mg of protein-1 (data not shown). Hydrolysis of (Ala)6 could not be detected in 200-fold-concentrated supernatant obtained from L. monocytogenes grown in DM (Fig. 2B). Similarly, peptidase activity towards valine-containing peptides (i.e., Val-Gly [n = 2], Val-Gly-Asp-Glu [n = 4], Val-Leu-Ser-Glu-Gly [n = 5], Arg-Val-Tyr-Ile-His-Pro-Phe [n = 7], Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr [n = 9], and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [n = 10]) could be detected only in CE (several breakdown products were found, including individual amino acids) and not in concentrated supernatant of L. monocytogenes (data not shown). The data indicate that the hydrolysis of oligopeptides occurs in the cytoplasm.


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  		FIG. 2.   HPLC analysis of hexa-alanine [(Ala)6] incubated with CE (A) and 200-fold-concentrated supernatant (B). CE (1.2 mg of protein ml-1) and 200-fold-concentrated supernatant (0.4 mg of protein ml-1) were incubated with 0.5 mM (Ala)6 in 50 mM potassium phosphate (pH 6.9) containing 5 mM MgSO4, and samples were taken and analyzed after derivatization with dansyl chloride by reversed-phase HPLC. ------, t = 0; ----, t = 40 min; ... ., t = 90 min. CE and concentrated supernatant were prepared from cells grown in DM at 30°C and harvested during exponential growth.

Peptidase activity in CE and 200-fold-concentrated supernatant of L. monocytogenes was also assayed by use of chromogenic substrates. A peptidase activity of 2 nmol min-1 mg of protein-1 was found in CE with succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitroanilide, whereas no release of nitroanilide could be detected upon incubation of CE with the other substrates (i.e., alanyl-prolyl-p-nitroanilide, glycyl-prolyl-p-nitroanilide, acyl-alanyl-alanyl-alanyl-p-nitroanilide, and isoleucyl-prolyl-arginyl-p-nitroanilide). None of the chromogenic substrates tested were hydrolyzed upon incubation with concentrated supernatant.

Oligopeptide transport in L. monocytogenes. Uptake of the pentapeptide Val-Leu-Ser-Glu-Gly and the hexapeptide (Ala)6 in cells of L. monocytogenes was investigated. Significant rates of uptake of both peptides (final concentration, 0.3 mM) were detected in cells incubated in 50 mM potassium phosphate (pH 6.9)-5 mM MgSO4 in the presence of glucose (Fig. 3). Under these conditions, a PMF of -130 mV and an intracellular ATP concentration of 7.5 mM were recorded (data not shown). The addition of the potassium ionophore valinomycin (1.5 µM) plus the potassium proton exchanger nigericin (2 µM), which was without effect on intracellular ATP levels but resulted in the complete dissipation of the PMF (data not shown), partly inhibited both Val-Leu-Ser-Glu-Gly and (Ala)6 uptake (Fig. 3). The uptake of the dipeptide Pro-Ala, which has been shown to proceed via a PMF-dependent carrier protein (51), was completely abolished upon addition of valinomycin and nigericin (Fig. 3C). At pH 6.0, the uptake rates of both oligopeptides were slightly decreased compared to the uptake rates at pH 6.9. The addition of both valinomycin and nigericin to the assay mixtures at pH 6.0 had a more dramatic effect on the transport rate of the oligopeptides than did addition at pH 6.9 (about 70% reduction compared to the control; data not shown), which is probably a result of lowering of the internal pH. These experiments show that Val-Leu-Ser-Glu-Gly and (Ala)6 transport can proceed in the absence of a PMF. The nature of the energy source for oligopeptide transport was further investigated by analysis of the effect of the phosphate analog vanadate on Val-Leu-Ser-Glu-Gly and (Ala)6 transport. Uptake of both peptides was completely inhibited in the presence of 0.2 mM vanadate in 50 mM K-HEPES (pH 7.5)-5 mM MgSO4 (Fig. 4). Under these conditions, both the PMF and the intracellular ATP concentration decreased to about 90% of their original values (data not shown). Vanadate had no influence on Pro-Ala uptake, which was expected, since dipeptide transport in L. monocytogenes is driven by the PMF (51) and is not affected by ATP directly. The results show that oligopeptide transport in L. monocytogenes proceeds via a transport system which is different from the di- and tripeptide transport system in the organism. The inhibition of oligopeptide transport by vanadate is most likely due to its specific interference with ATP-dependent activation of the transporter, as has been demonstrated before for several other ATP-dependent transporters (12, 25, 39, 50).


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  		FIG. 3.   Uptake of peptides in L. monocytogenes. Uptake assays with Val-Leu-Ser-Glu-Gly (A), (Ala)6 (B), and Pro-Ala (C) were performed in 50 mM potassium phosphate (pH 6.9) containing 5 mM MgSO4 in glucose-energized cells in the absence (closed symbols) and presence (open symbols) of nigericin (2 µM) and valinomycin (1.5 µM).


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  		FIG. 4.   Uptake of peptides in L. monocytogenes. Uptake assays with Val-Leu-Ser-Glu-Gly (A), (Ala)6 (B), and Pro-Ala (C) were performed in 50 mM potassium-HEPES (pH 7.5) containing 5 mM MgSO4 in glucose-energized cells in the absence (closed symbols) and presence (open symbols) of vanadate (0.2 mM).

Effect of proteolytic enzymes and bacteria on growth of L. monocytogenes. As anticipated, L. monocytogenes failed to grow in DM lacking amino acids or in DM lacking amino acids with beta -casein or Na-caseinate present as the sole source of nitrogen. The addition of beta -casein or Na-caseinate which had been hydrolyzed with a protease from B. licheniformis to DM without amino acids resulted in a stimulation of growth of L. monocytogenes. With hydrolyzed Na-caseinate (containing alpha , beta , and kappa  casein), an OD620 of approximately 0.55 was reached in 30 h, whereas with hydrolyzed beta -casein the OD620 was 0.45 (data not shown).

Growth of L. monocytogenes at 20°C in DM with Na-caseinate present as the sole source of nitrogen was stimulated in the presence of B. cereus or P. fragi. These bacteria produced large clearing zones of 5.0 and 4.5 mm, respectively, in skim milk agar plates (data not shown), indicating their capability to degrade casein. In addition, in the TCA-soluble-peptide assay, the absorbance at 275 nm increased from 0.2 to 2.2 for B. cereus cells and from 0.2 to 1.9 for P. fragi cells, indicating significant proteolytic degradation of casein (data not shown). In the control experiment, L. monocytogenes increased from 1.0 × 106 to 1.6 × 107 CFU/ml in 75 h, whereas with either B. cereus or P. fragi, L. monocytogenes grew to about 1.0 × 109 CFU/ml. The growth of B. cereus or P. fragi was not affected by the presence of L. monocytogenes; levels amounted to 1.3 × 107 and 5.0 × 107 CFU/ml, respectively (data not shown). Comparable numbers for growth of these organisms have been reported previously (35). Likewise, L. monocytogenes which had been pregrown with B. cereus or P. fragi as described in Materials and Methods reached high levels (from 5.8 × 105 to about 2 × 109 CFU/ml at 30°C in 25 h) in DM without amino acids containing Na-caseinate, whereas in the control experiment L. monocytogenes grew only up to 6.3 × 106 CFU/ml (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In a previous study, we showed that L. monocytogenes takes up di- and tripeptides via a PMF-dependent permease that can supply the pathogen with amino acids essential for growth (51). The present results show that L. monocytogenes is, in addition, able to grow on oligopeptides as a source of essential amino acids, and the experiments reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation.

The growth experiments with DM where valine was replaced by valine-containing peptides of various lengths suggest that the oligopeptide uptake system transports peptides containing up to eight amino acid residues. The translocation of the peptide seems to be the limiting step for utilization, since the valine-containing nona- and decapeptides, which cannot serve as sources of amino acids essential for growth (Fig. 1), were found to be hydrolyzed upon incubation with CE from L. monocytogenes. However, since only a few oligopeptides were tested, it is possible that the transportable species can also be longer than 8 residues. The oligopeptide permeases (Opp) of gram-negative bacteria (e.g., Escherichia coli and Salmonella typhimurium) transport peptides up to and including hexapeptides. Transport of longer peptides in gram-negative bacteria may be restricted by the upper size exclusion limits of the outer membrane pores rather than the transporter (37). Indeed, in gram-positive bacteria, the size restriction seems to be more variable. In Bacillus subtilis, tri-, tetra-, and pentapeptides can be transported via two different oligopeptide transporters (22, 38). Streptococcus pneumoniae possesses an oligopeptide permease that functions in the uptake of peptides consisting of 2 to 7 residues (3), and recently a hexa- heptapeptide permease in Streptococcus gordonii was identified (17). Lactococcus lactis expresses an Opp that is capable of transporting peptides of 4 to 8 residues (25, 49). However, recent experiments, in which translocation of oligopeptides formed by the action of the cell-wall-bound extracellular proteinase (PrtP) on the natural substrate beta -casein (instead of commercially available peptides) was analyzed, indicate that oligopeptides consisting of up to 10 amino acids may be transported by L. lactis (26).

All oligopeptide transport systems described to date belong to the family of binding-protein-dependent transport systems that are composed of multiple subunits and use ATP or a related energy-rich, phosphorylated intermediate to drive the peptide uptake (37). The finding that oligopeptide transport in L. monocytogenes is specifically inhibited by vanadate whereas the PMF (i.e. glycolysis) and the ATP production are not inhibited under these conditions indicates that the energy requirement for oligopeptide transport is not supplied by the PMF. The inhibition by vanadate may be attributed to its direct interference with ATP-dependent activation of the transporter, as was suggested for other ATP-dependent transport systems (12, 25, 39, 50). Therefore, the Opp of L. monocytogenes most likely also belongs to the family of binding-protein-dependent transporters. The partial inhibition of Val-Leu-Ser-Glu-Gly and (Ala)6 uptake as a result of the dissipation of the PMF by adding nigericin plus valinomycin is probably a secondary effect since PMF dissipation may coincide with changes in internal pH, ATP pools, and turgor pressure (1, 25, 50). In contrast, uptake of the dipeptide Pro-Ala in L. monocytogenes, which is driven by the PMF (51), was completely inhibited by the combination of the two ionophores whereas vanadate had no effect on Pro-Ala transport (Fig. 3 and 4).

The oligopeptide transport system of L. monocytogenes has a relatively high level of activity. The observed rates of Val-Leu-Ser-Glu-Gly and (Ala)6 uptake at pH 6.9 (at an external peptide concentration of 0.3 mM) in cells grown in BHI were approximately 35 and 60 nmol min-1 mg of protein-1, respectively, whereas Pro-Ala is transported at a rate of about 15 nmol min-1 mg of protein-1 under the same conditions (Fig. 3). In L. lactis cells cultivated in DeMan, Rogosa, and Sharpe (MRS) broth, tetra-, penta-, and hexa-alanine are transported with rates of 2.3, 8.0, and 2.3 nmol min-1 mg of protein-1, respectively, at an external peptide concentration of 0.5 mM (25). Rates of uptake of zwitterionic di- and tripeptides in L. monocytogenes and L. lactis have been shown to be of the same magnitude (15, 25, 45, 51). Since L. monocytogenes cannot utilize proteins as a source of amino acids, the relatively high rates of oligopeptide uptake can be advantageous to the organism during its growth in foods that are deficient in free amino acids and small peptides but rich in oligopeptides as a result of proteolytic activity of other microorganisms (see also below).

For L. lactis, more than 10 peptidases displaying different substrate specificities have been identified over the years (26, 33), whereas for L. monocytogenes this area of research is almost completely unexploited. In our previous study, we detected N-terminal aminopeptidase activities in CE using lysyl-p-nitroanilide, leucyl-p-nitroanilide, and alanyl-p-nitroanilide. These activities were between 0.2 and 0.8 nmol min-1 mg of protein-1 and are about 50- to 100-fold lower than those reported for lactococci (5, 46, 51). In the present work, release of nitroanilide in CE could not be demonstrated with the chromogenic substrates alanyl-prolyl-p-nitroanilide, glycyl-prolyl-p-nitroanilide, acyl-alanyl-alanyl-alanyl-p-nitroanilide, and isoleucyl-prolyl-arginyl-p-nitroanilide, whereas a peptidase activity of 2 nmol min-1 mg of protein-1 was found in CE with succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitroanilide. A similar hydrolysis rate (2 nmol min-1 mg of protein-1) could be observed with (Ala)6 as a substrate. Tan and Konings (46) reported that the aminopeptidase N (PepN) of L. lactis, which shows high activity towards lysyl-p-nitroanilide, had very low activity towards the chromogenic substrates alanyl-prolyl-p-nitroanilide and alanyl-alanyl-alanyl-p-nitroanilide. Considering the relatively low aminopeptidase activity found for L. monocytogenes compared to that of L. lactis, this would validate our present findings. To degrade proline-containing oligopeptides, lactic acid bacteria generally make use of an X-prolyl-dipeptidyl-peptidase (PepXP) (8). With the chromogenic substrate glycyl-prolyl-p-nitroanilide, specific PepXP activities between 85 and 300 nmol min-1 mg of protein-1 have been recorded for L. lactis (7, 20, 31). However, with L. monocytogenes no release of nitroanilide was found with glycyl-prolyl-p-nitroanilide, suggesting a low level of PepXP-like activity. This in turn would explain the accumulation particularly of proline-containing peptides in cells of L. monocytogenes after growth in peptone or in defined medium in the presence of those peptides (4).

Products that have been involved in food-borne listeriosis (raw milk, raw meat, and minimally processed vegetables) generally harbor diverse populations of microorganisms. These include, besides L. monocytogenes, proteolytic microbes like Pseudomonas spp., Bacillus spp., and lactic acid bacteria. In addition, L. monocytogenes and Pseudomonas spp. appear frequently in pasteurized dairy products as postprocessing contaminants, whereas spores of bacilli survive pasteurization (2, 9, 11, 16, 23, 30, 34). In this study, P. fragi DSM 3456 and B. cereus VC2 were shown to enhance the growth of L. monocytogenes in a medium with casein present as the sole amino acid source. In addition, hydrolysis of casein by using purified B. licheniformis protease increased the growth of L. monocytogenes. These results suggest that proteolysis of the milk protein casein can provide stimulatory factors (i.e., large and small peptides and amino acids) for the growth of L. monocytogenes (21, 51). The oligopeptide transport system of L. monocytogenes described herein might have been of importance in the utilization of these breakdown products. Taking into account the psychrotrophic nature of L. monocytogenes, psychrotrophic species of Pseudomonas and Bacillus may especially stimulate the growth of L. monocytogenes during refrigerated storage of foods (9, 47). In fermented dairy products such as cheese, casein is degraded by the cell-envelope-located proteinase (PrtP) of lactococci, which results in the formation of more than 100 different peptides ranging from 4 to 10 residues (18). To date, no significant release of amino acids or di- and tripeptides has been observed to occur in hydrolysates formed by various lactococcal proteinases (26). In the initial fermentation phase (high pH, low level of lactic acid), the oligopeptide transport system may be crucial to supply L. monocytogenes with essential amino acids allowing it to grow. Recently, L. monocytogenes has been shown to exhibit an adaptive acid tolerance response (ATR) following adaptation to mildly acidic conditions; this response is capable of protecting cells from normally lethal acid stress (24, 36). The oligopeptide transport system of the pathogen in combination with the development of ATR might therefore result in increased numbers of L. monocytogenes cells in fermented milk products (14).

    ACKNOWLEDGMENT

This research was financially supported by the European Community, contract EC-AIR1-CT92-0125.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Food Science, Food Chemistry/Microbiology Section, Agricultural University Wageningen, Bomenweg 2, 6703 HD Wageningen, The Netherlands. Phone: 31317484981. Fax: 31317484893. E-mail: tjakko.abee{at}algemeen.lenm.wau.nl.

dagger Present address: Netherlands Institute for Dairy Research, 6710 BA Ede, The Netherlands.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Appl Environ Microbiol, March 1998, p. 1059-1065, Vol. 64, No. 3
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