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Applied and Environmental Microbiology, February 2002, p. 813-819, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.813-819.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of Glycine Betaine Porter I from Listeria monocytogenes and Its Roles in Salt and Chill Tolerance

Mary Lou Mendum and Linda Tombras Smith^*

Department of Agronomy and Range Science, University of California, Davis, California 95616

Received 20 September 2001/ Accepted 26 November 2001

Top Abstract Introduction Materials and Methods Results Discussion References

 
Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na⁺-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and -butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Listeria monocytogenes is a food-borne pathogen responsible for a variety of infective syndromes in humans, including meningitis, encephalitis, septicemia, and spontaneous abortion (3). Listeriosis can be very serious in certain high-risk groups, such as individuals who have an underlying condition leading to the suppression of T-cell-mediated immunity. Fatality rates worldwide have been reported to be as high as 34% (3).

Conditions of osmotic stress and low temperature favor the growth of the otherwise poor competitor L. monocytogenes over that of other bacteria in a mixed culture. The ability of L. monocytogenes to grow under conditions of high osmolarity and low temperature has been well documented (1, 3, 17). One primary mechanism by which eubacteria adapt to osmotic stress is the accumulation of compatible solutes called osmolytes: low-molecular-weight organic compounds that have comparatively minimal deleterious effects on cell biochemistry at high concentrations (18).

Glycine betaine (N,N,N-trimethylglycine) is a highly effective osmolyte in many gram-negative and gram-positive bacteria, including L. monocytogenes (2, 5, 7, 13). This solute has a number of chemical properties that make it useful as a protectant against osmotic stress, including high solubility. Thus, it can accumulate to high concentrations in the cytoplasm without producing adverse effects on protein structure, protein-protein interactions, enzyme-substrate interactions, or protein-nucleic acid interactions, as would high concentrations of inorganic salts (18). The intracellular accumulation of this osmolyte in bacterial cells can occur either by de novo synthesis or by transport from the growth medium.

In addition to osmotolerance, L. monocytogenes has the unusual property of utilizing glycine betaine for cryotolerance (7). In response to either stress, glycine betaine is accumulated in this pathogen solely from the growth medium, via active transport. L. monocytogenes has two specific permeases devoted to glycine betaine transport. One is an ATP-dependent transporter, glycine betaine porter II, which has been characterized biochemically and genetically (6, 7). The other permease, glycine betaine porter I, is an Na⁺-betaine symporter. This porter was identified in vesicles (4), and the gene presumed to encode this transport system has been identified and its nucleotide sequence has been reported (11). However, little is known about the properties of glycine betaine porter I in whole cells.

The work reported here characterizes glycine betaine porter I activity in whole cells, including the efficacy of various glycine betaine analogs as inhibitors of betaine uptake. The usefulness of these analogs in reducing the growth of L. monocytogenes under salt-stressed conditions was also determined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and reagents.
L. monocytogenes strains used in this work were DP-L1044 (hly::Tn917-LTV3) (15), a nonhemolytic mutant lacking hemolysin A; and LTG59 (gbu::Tn917-LTV3), which lacks glycine betaine porter II activity (6). Both are derivatives of the wild-type isolate 10403S (15). Cultures were maintained on solid brain heart infusion (BHI) containing 10 µg of chloramphenicol/ml. Modified Pine's medium (9, 10), which contained 0.5% glucose but no choline, was used as a defined medium. [methyl-¹⁴C]glycine betaine was prepared enzymatically by the oxidation of [methyl-¹⁴C]choline (NEN Research Products) (8). Choline oxidase, sarcosine, dimethylglycine, glycine betaine, trigonelline, alanine, proline, choline, carnitine, glycine, -aminobutyric acid, and ß-alanine were purchased from Sigma. N-Methylproline and -butyrobetaine were purchased from Aldrich. Triethylglycine was a generous gift from the laboratory of Gary M. Smith, Department of Food Science and Technology, University of California at Davis.

Measurement of glycine betaine uptake rates in salt- and cold-adapted bacteria.
L. monocytogenes cultures grown in BHI broth were centrifuged at 5,000 x g for 10 min, resuspended in modified Pine's medium, and used to inoculate (1%) modified Pine's medium containing NaCl or other additions as specified for individual experiments. These cultures were grown to late log phase at the indicated temperatures. Grown cultures were centrifuged and resuspended in an assay buffer at pH 7, which contained 22 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES buffer), 27 mM K₂HPO₄, 1 mM Na₂SO₄, 5 mM MgSO₄, 5% glucose, and NaCl or other additives as required for individual experiments. Cultures were assayed directly for chill stress experiments or were diluted fivefold with additional assay buffer for salt stress experiments. After a 30-min incubation at the assay temperature, uptake of [¹⁴C]glycine betaine (0.02 to 0.05 µCi) was determined using the method of Ko et al. (7). Typically, 100 µM glycine betaine was used for transport assays, and uptake rates were normalized to total cellular protein, as determined by the Pierce BCA Protein Assay kit (14). The osmotic strength of the media was directly measured using a Wescor, Inc. 5100C Vapor Pressure Osmometer. The uptake rates reported here are the average of from three to five replicates, and the error was generally about 5%.

Glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake in the presence of 4% NaCl at 30°C and in the absence of salt at 7°C. For osmotic upshock experiments, cells were incubated in assay buffer without salt for 30 min and were then diluted fivefold into test tubes containing assay buffer and NaCl (final concentration, 4%). Radioactive glycine betaine was added to successive tubes of bacteria at 8-min intervals, and its uptake was determined as described above.

For preloading experiments, cultures diluted in assay buffer and 4% NaCl were distributed into several assay tubes and were incubated for 30 min at the assay temperature, and then 5 mM glycine betaine analog was added to each assay tube. Controls were incubated without added glycine betaine or analogs. The [¹⁴C]glycine betaine (final concentration, 100 µM) was then added successively to each of the tubes at 8-min intervals to initiate the assay. For samples preloaded with 5 mM glycine betaine, transport rates were calculated taking into account the dilution of the radioactive label.

NMR spectral analysis of cellular extracts.
Cellular extracts from cultures grown in modified Pine's medium containing 4% NaCl and 5 mM betaine analog were subjected to natural abundance ¹³C nuclear magnetic resonance (NMR) spectroscopy as previously described (7). The identity of osmolytes was determined by comparison to known standards.

Growth measurements.
Bacteria were grown in BHI broth overnight and were then centrifuged and resuspended in modified Pine's medium without salt. The resuspension was used to inoculate tubes of modified Pine's medium with additions of NaCl, glycine betaine, and glycine betaine analogs as described. Growth was monitored by measuring turbidity as previously described (7). Mean doubling times of at least three separate experiments are reported.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Salt-activated glycine betaine uptake.
Strain DP-L1044 possesses both of the glycine betaine transporters that are in wild-type L. monocytogenes: the ATP-driven transporter, glycine betaine porter II (6); and the Na⁺-betaine symporter, glycine betaine porter I (4). In strain LTG59, the gene for porter II has been interrupted by Tn917-LTV3, leaving only porter I active (6). While the focus of this work is the symporter, the properties of the wild-type strain were also determined for comparison purposes. Both strains showed maximum glycine betaine uptake at 4% NaCl. However, strain DP-L1044 accumulated glycine betaine more rapidly than did strain LTG59 at all concentrations of salt tested (Fig. 1). The difference between the two strains was moderate below 3% NaCl, but when assayed at or above 4% NaCl, strain DP-L1044 accumulated glycine betaine at a rate over five times that of strain LTG59. Furthermore, at NaCl concentrations above 6%, glycine betaine uptake was not observed in strain LTG59. These results indicate the predominance of the ATP-driven transporter in salt-activated glycine betaine uptake, particularly at a higher NaCl concentration.

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FIG. 1. Salt-dependent glycine betaine uptake in L. monocytogenes. Cultures of strains DP-L1044 (•) and LTG59 () were grown at 30°C in Pine's medium containing the indicated concentrations of NaCl. The cells were centrifuged, resuspended in buffer containing the same NaCl concentrations, and then assayed for glycine betaine uptake. The ranges of replicate values are indicated by the error bars.

Alternative osmotic stressors and glycine betaine uptake.
To determine whether glycine betaine uptake was a specific response to NaCl or was a general response to osmotic stress, the ability of alternative osmotic stressors to stimulate glycine betaine uptake was tested. Bacteria were grown in modified Pine's medium to which either 4% NaCl or an osmotically equivalent concentration of other salts had been added. In all media tested, glycine betaine uptake was observed in both strains, and in most media, DP-L1044 took up glycine betaine significantly faster than did LTG59 (Fig. 2). Phosphate salts were least effective in activating betaine transport in both strains but to different extents. For example, when phosphate ion replaced chloride ion, the rate of uptake decreased twofold for LTG59 but over sevenfold for DP-L1044. This differential decrease in transport may be due to competitive inhibition of the ATP-dependent transporter by phosphate ion at the site of ATP binding. When an osmotically equivalent concentration of K⁺ replaced Na⁺, glycine betaine uptake was depressed in LTG59, indicating that the low level of Na⁺ present in the growth and assay medium was not adequate to allow efficient uptake of glycine betaine via glycine betaine porter I of LTG59. Taken together, these results show that the ATP-driven transporter is osmotically activated, not requiring specific cations or anions for activity, and that the symporter requires Na⁺ but not Cl^- for efficient uptake of glycine betaine.

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FIG. 2. Alternative osmotic stressors and glycine betaine uptake in L. monocytogenes. Cultures of strains DP-L1044 (filled bars) and LTG59 (open bars) were grown at 30°C in Pine's medium containing the indicated concentrations of NaCl or other osmotic stressors. The cells were centrifuged, resuspended in buffer containing the same osmotic stressor, and then assayed for glycine betaine uptake. The ranges of replicate values are indicated by the error bars. sat'd, saturated.

Glycine betaine uptake after osmotic upshock.
To determine the role of each glycine betaine transporter in osmotic adaptation after osmotic upshock, bacteria grown in medium without salt were subjected to 4% NaCl and a time course of glycine betaine uptake was determined (Fig. 3).

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FIG. 3. Osmotic upshock and the onset of glycine betaine uptake. Cultures of strains DP-L1044 (•) and LTG59 () were grown at 30°C in Pine's medium without added salt. The cells were centrifuged, resuspended in assay buffer without salt, and then diluted with buffer containing NaCl to a final concentration of 4% NaCl, as described in Materials and Methods. Glycine betaine uptake was measured at the indicated intervals after upshock. The ranges of replicate values are indicated by the error bars.

Strain LTG59 showed an essentially instantaneous glycine betaine uptake rate of about 2 nmol/min/mg of protein, followed by a gradual increase in activity to about 4 nmol/min/mg of protein. There was little further increase in uptake, even after 4 h. In contrast, the betaine uptake activity in DP-L1044, which was similar to that of LTG59 immediately after upshock, increased to more than 12 nmol/min/mg of protein 4 h after upshock. This result argues for the dominance of the ATP-driven transporter in long-term osmotic adjustment.

Cold-activated glycine betaine uptake.
Osmolytes have been shown to confer cryotolerance to L. monocytogenes, a pathogen known for its ability to grow actively at refrigerator temperatures. To determine the relative role of the symporter in cryotolerance, the temperature dependence of uptake activity in both strains was determined (Fig. 4). Both strains showed maximum glycine betaine uptake rates at 12°C. Over most of the temperature range tested, strain DP-L1044 took up glycine betaine at a rate approximately 10 times that of strain LTG59. These results suggest that the ATP-dependent transporter plays a dominant role in cold-induced glycine betaine uptake over the entire range of temperature tested.

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FIG. 4. Temperature-dependent glycine betaine uptake in L. monocytogenes. Cultures of strains DP-L1044 (•) and LTG59 () were grown at the indicated temperatures in Pine's medium without salt. The cells were centrifuged, resuspended in buffer without salt, and assayed at the same temperatures for glycine betaine uptake. The ranges of replicate values are indicated by the error bars.

Inhibition of glycine betaine uptake.
Since glycine betaine uptake is a key element in the ability of L. monocytogenes to grow under environmentally stressful conditions, it was of interest to determine if substrate analogs of glycine betaine could inhibit glycine betaine uptake and thus growth as well. Eleven structural analogs of glycine betaine (Fig. 5) were tested for their ability to inhibit osmotic and chill-activated glycine betaine transport in both strains (Table 1). Of the compounds tested, the amino acids had the least effect on glycine betaine uptake rates. Proline had a small inhibitory effect in both strains over the longer time span required to measure cold-induced uptake. The proline analog N-methylproline reduced salt-activated glycine betaine uptake somewhat more effectively in strain LTG59 but was less effective than proline at reducing uptake in DP-L1044 and cold-stressed LTG59 cultures. The amino acid -aminobutyric acid reduced cold-activated glycine betaine uptake in strain LTG59 only.

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FIG. 5. Structures of glycine betaine and its analogs were tested for their ability to inhibit glycine betaine uptake by L. monocytogenes. The 12 analogs were selected on the basis of their structural similarity to glycine betaine or on their ability to act as osmolytes in other bacteria.

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TABLE 1. Inhibition of glycine betaine transport by betaine analogs and related compounds

Sarcosine (monomethylglycine) increased salt-activated but not cold-activated glycine betaine uptake in strain DP-L1044 and cold-activated but not salt-activated uptake in LTG59. Dimethylglycine was not an effective inhibitor of uptake in strain DP-L1044. However, uptake by strain LTG59 showed a similar but smaller increase in cold-activated uptake than that caused by sarcosine. Dimethylglycine reduced glycine betaine uptake by about 15% in salt-stressed LTG59 cells.

Carnitine shares structural similarities with glycine betaine, but it is also an excellent osmoprotectant in L. monocytogenes (12). Carnitine and its biosynthetic precursor -butyrobetaine inhibited cold-activated glycine betaine uptake in both strains. In addition, carnitine inhibited salt-activated glycine betaine uptake in DP-L1044 but not in LTG59, suggesting that carnitine might act as an inhibitor of the ATP-dependent transporter only.

Choline, which is a precursor to glycine betaine in many bacterial species, had no effect on glycine betaine uptake in strain DP-L1044 but did inhibit cold-activated uptake by about 50% in strain LTG59, suggesting that it affects the Na⁺-betaine symporter.

Trigonelline, on the other hand, was able to reduce glycine betaine uptake in strain LTG59 by two-thirds under salt-stressed conditions and by almost as much in the cold. Strain DP-L1044 showed a small reduction under salt stress, while cold-activated uptake was not affected. These data are consistent with trigonelline's being a potent and specific inhibitor of glycine betaine uptake through glycine betaine porter I, while leaving the ATP-driven transporter unaffected. Because the rate of cold-activated transport in strain LTG59 was exceedingly low, its properties were not further investigated.

Glycine betaine transport in cells preincubated with inhibitors.
While a number of substrate analogs showed inhibition of uptake activity (Table 1), the mechanism by which these compounds act may be strikingly different. Two possibilities are kinetic inhibition (competitive or otherwise) at the site of transport and inhibition caused by downregulation of the porter activity. The latter would occur, for example, if the inhibitor is transported and accumulated in the cell to significant levels, thus alleviating the requirement for subsequent accumulation of osmolytes. In the latter mechanism, the level of inhibition would increase with time as the intracellular concentration of inhibitor increases. To shed light on the mechanism(s) of inhibition caused by the betaine analogs, cells were incubated with 5 mM inhibitor for various lengths of time, and then [¹⁴C]glycine betaine was added and transport activity was assayed (Fig. 6). By varying the preincubation time before glycine betaine uptake was measured, the time dependence of inhibition could be determined.

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FIG. 6. Inhibition of glycine betaine transport after preloading with glycine betaine analogs and related compounds. Cultures of strains DP-L1044 (circles) and LTG59 (squares) were grown in modified Pine's medium with 4% NaCl. The cells were centrifuged and resuspended in buffer with salt, and glycine betaine analogs were added to a final concentration of 5 mM. Controls were incubated without glycine betaine or analog. Uptake of [methyl-14C]glycine betaine was measured at 8-min intervals after the addition of analog as described in Materials and Methods. The first time point was 4 min after the addition of analog. (A) Glycine betaine uptake after preloading with 5 mM cold glycine betaine (filled symbols) or with 5 mM choline (open symbols). (B) Glycine betaine uptake after preloading with 5 mM sarcosine (filled symbols) or with 5 mM dimethylglycine (open symbols). (C) Glycine betaine uptake after preloading with 5 mM carnitine (filled symbols) or with 5 mM -butyrobetaine (open symbols). (D) Glycine betaine uptake after preloading with 5 mM trigonelline (filled symbols) or with 5 mM triethylglycine (open symbols). Glycine betaine uptake is expressed as a percentage of the control rates. The ranges of replicate values are indicated by the error bars.

Preincubation with the amino acids and amino acid analogs (glycine, alanine, proline, N-methylproline, -aminobutyric acid, and ß-alanine) had little effect on glycine betaine uptake with either strain of L. monocytogenes (data not shown). Choline and sarcosine stimulated glycine betaine uptake over the first 20 min of preincubation (Fig. 6A and B). There was little effect on glycine betaine uptake after 60 min of preincubation with either of these compounds. A transitory stimulation of labeled glycine betaine uptake was also observed when cells were preincubated with unlabeled glycine betaine (Fig. 6A). However, an inhibition of betaine uptake in strain DP-L1044 was observed after preincubation with dimethylglycine, carnitine, or -butyrobetaine (Fig. 6B and C). These compounds had little effect on glycine betaine uptake in strain LTG59. This result suggests that dimethylglycine, carnitine, and -butyrobetaine inhibit glycine betaine uptake primarily through the ATP-dependent transporter.

The glycine betaine analogs trigonelline and triethylglycine inhibited uptake in strain DP-L1044 only slightly. In contrast, these analogs inhibited glycine betaine uptake in strain LTG59 by about 70% over the entire course of the experiment (Fig. 6D), suggesting that they function as competitive inhibitors of glycine betaine porter I.

Growth of L. monocytogenes in the presence of glycine betaine uptake inhibitors.
The five glycine betaine analogs which reduced salt-activated glycine betaine uptake in the inhibition and preloading experiments were further examined for their effect on the growth of L. monocytogenes under high-salt conditions. The structural similarity which allows these compounds to inhibit glycine betaine uptake could also allow them to be transported into the cell via the glycine betaine uptake systems, and if accumulated, they could act as osmoprotectants as well. To examine this possibility, strain DP-L1044 was grown in modified Pine's medium containing NaCl and the analogs as indicated (Table 2). Under these conditions, the doubling time was 7.4 h in the presence of 100 µM glycine betaine and 30 h if no osmoprotectant was present in the medium. Bacteria grown in medium containing carnitine had a doubling time of 9.8 h, which shows that this compound is not quite as effective an osmoprotectant as glycine betaine. Dimethylglycine, -butyrobetaine, and triethylglycine were also osmoprotective but were not as effective as either glycine betaine or carnitine. NMR analysis of cell extracts verified that carnitine, dimethylglycine, -butyrobetaine, and triethylglycine were indeed accumulated to significant levels by osmotically stressed L. monocytogenes cultures (data not shown). In contrast, cultures grown in trigonelline had a doubling time of 65 h, a twofold increase over the doubling time of the no-addition control. Also, NMR analysis indicated that trigonelline is not accumulated by L. monocytogenes (data not shown). While the reason for this inhibition is unclear, trigonelline did not affect growth of strain DP-L1044 in the absence of salt stress (data not shown), suggesting that its effect may be limited to osmotically stressful conditions.

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TABLE 2. Growth enhancement of L. monocytogenes DP-L1044 by glycine betaine or its analogs

Growth of L. monocytogenes in the presence of glycine betaine uptake inhibitors and glycine betaine.
Since fresh and processed meats and dairy products contain glycine betaine in the micromolar range (12), it was of interest to determine the inhibitory effect of betaine analogs on growth in the presence of glycine betaine (Table 3). Under these conditions, the growth of both strains was appreciably inhibited by triethylglycine. Also, the effect of amending the growth medium with two inhibitors in combination was not significantly different from the results of each inhibitor added singly (data not shown). These results suggests that inhibiting glycine betaine uptake can produce a measurable reduction in the growth rate of L. monocytogenes, even in the presence of physiological concentrations of glycine betaine.

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TABLE 3. Effect of glycine betaine inhibitors on growth of salt-stressed L. monocytogenes in the presence of glycine betaine

Top Abstract Introduction Materials and Methods Results Discussion References

 
The two glycine betaine transporters of L. monocytogenes provide effective protection against osmotic stress when glycine betaine is available in the growth medium. Results reported here demonstrate that both glycine betaine porter I and glycine betaine porter II are osmotically activated (Fig. 1 and 3). However, the biochemical properties of these transport systems differ significantly. For example, salt-activated porter I activity appeared to be lower than that of porter II. On the other hand, glycine betaine porter I appeared to be responsible for the majority of the glycine betaine uptake immediately following osmotic upshock. The differences between the two transport systems illustrate the unique role that each porter occupies in osmotic adaptation. Glycine betaine porter I responds quickly to osmotic upshock to provide immediate protection, but it can also provide long-term protection for low levels of stress. That porter I is inactive in the absence of Na⁺ is probably not of physiological significance, since the complete absence of Na⁺ is likely not to be encountered in nature. The ATP-dependent transporter, porter II, plays a dominant role in long-term adaptation, particularly with high levels of salt in the growth medium. Under chill stress conditions, however, glycine betaine uptake occurred primarily through the ATP-dependent transporter, with glycine betaine porter I playing a relatively minor role. This result, which was obtained using whole cells, is not inconsistent with earlier work on membrane vesicles, in which porter I showed negligible activity in response to chill stress (4).

In addition to glycine betaine, carnitine and dimethylglycine are accumulated by L. monocytogenes and act as stress protectants. Regulation of the accumulation of the three osmolytes appears to be interdependent in the wild-type strain DP-L1044, as preloading with carnitine or dimethylglycine reduces the rate of glycine betaine uptake (Fig. 6B and C). These results are consistent with those from a previous report showing that glycine betaine uptake can be inhibited by carnitine (16). However, while we observed significant inhibition by carnitine and dimethylglycine in strain DP-L1044, we did not in strain LTG59, which lacks porter II. Moreover, the results given in Fig. 6 suggest multiple mechanisms of inhibition. For example, carnitine and dimethylglycine inhibited transport in strain DP-L1044 but not in strain LTG59. On the other hand, -butyrobetaine inhibited uptake in both strains, while triethylglycine and trigonelline were effective inhibitors of transport in LTG59 but not in DP-L1044. These varied responses serve to highlight the complexity of the regulation of glycine betaine transport. For instance, several of these analogs were shown to accumulate in the cell and function as osmolytes, thus restoring turgor pressure and relieving the requirement for additional osmolytes. Evidence for this possibility is that the extent of time-dependent inhibition by these analogs roughly correlates with their ability to function as osmotic stress protectants (Table 2). In contrast, the effective inhibitors of LTG59, triethylglycine and trigonelline, show no time dependence, suggesting that these may function as competitive inhibitors at the site of transport. Additional support for this suggestion is that the accumulation of trigonelline could not be observed in osmotically stressed cultures. Triethylglycine, however, can be accumulated under these conditions.

Because glycine betaine occurs in many foods, inhibiting its uptake might be an effective way to slow the growth of salt-tolerant, food-borne pathogenic bacteria, such as L. monocytogenes, and thus reduce the incidence of food poisoning. While several glycine betaine analogs inhibited porter I activity, relatively little inhibition was observed with the wild-type strain. This result suggests that glycine betaine porter II exhibits more substrate specificity than the symporter, which is not surprising for a binding protein-dependent transporter such as porter II. Interestingly triethylglycine, which inhibited porter I activity to the greatest degree, was the only analog that also inhibited growth of both strains DP-L1044 and LTG59 in the presence of glycine betaine and elevated osmolarity. Since triethylglycine can be accumulated by L. monocytogenes, inhibition of growth by this analog may be explained by the fact that it interferes with either the ability of the cell to accumulate glycine betaine (downregulation of transport activity) or the ability of the accumulated glycine betaine to effectively protect the cell. In any case, this is a striking example of an additional strategy for inhibiting the growth of L. monocytogenes: not only extracellularly by kinetic inhibition of transport of osmolytes but also intracellularly by other mechanisms. Trigonelline, on the other hand, is not accumulated intracellularly and appreciably inhibits only glycine betaine porter I. Thus, no effect on growth rate would be expected in salt-stressed, wild-type cultures grown with glycine betaine, and none was observed. However, trigonelline appears to have a secondary effect, since it inhibits the growth of salt-stressed cells in the absence of added glycine betaine. The mechanism of this effect is presently under investigation.

The high affinity of both glycine betaine porters I and II allows L. monocytogenes to scavenge sufficient amounts of this compound for protection against osmotic stress, even when the concentration of glycine betaine in the growth medium is very low. Where glycine betaine is lacking, growth is inhibited. Our finding that a 20-fold excess of triethylglycine significantly decreases the growth rate of wild-type L. monocytogenes in the presence of glycine betaine demonstrates that even partial inhibition of glycine betaine uptake can have a significant effect on salt tolerance. Hence, design of effective inhibitors, which function either intra- or extracellularly, could be a viable approach in thwarting this pathogen's ability to survive osmotic and chill stresses.

 
We thank Gary Smith and his student Patrick Fitzgerald, both of the Food Science and Technology Department at the University of California, Davis, for the generous gift of triethylglycine.

We also acknowledge the support of Dairy Management Inc. Grant 98TSL-01.

 
* Corresponding author. Mailing address: Department of Agronomy and Range Science, University of California, Davis, CA 95616. Phone: (530) 752-6161. Fax: (530) 752-4361. E-mail: lsmith{at}ucdavis.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, February 2002, p. 813-819, Vol. 68, No. 2
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.2.813-819.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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