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Applied and Environmental Microbiology, April 2004, p. 2013-2020, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2013-2020.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Protective Effect of Sucrose and Sodium Chloride for Lactococcus lactis during Sublethal and Lethal High-Pressure Treatments

Adriana Molina-Höppner,1 Wolfgang Doster,2 Rudi F. Vogel,1 and Michael G. Gänzle*

Lehrstuhl für Technische Mikrobiologie, Technische Universität München, D-85350 Freising,1 Lehrstuhl E13, Fakultät für Physik, Technische Universität München, 85748 Garching, Germany2

Received 16 June 2003/ Accepted 20 December 2003


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ABSTRACT
 
The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity. To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes, Lactococcus lactis was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl. Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH ({Delta}pH), and multiple-drug-resistance (MDR) transport activity. Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment. In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by L. lactis; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death. The transmembrane {Delta}pH was reversibly dissipated during pressure treatment in any buffer system. Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity. In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure. These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components. The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress. Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.


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INTRODUCTION
 
The accumulation of compatible solutes, such as betaines, proline, and sugars, is a widespread response of microorganisms to elevated environmental osmotic pressures (41). According to a recent definition, a compatible solute is a "cytoplasmic cosolvent (solute) whose levels can be modulated over a broad range without disrupting cellular function" (24, 43). Compatible solutes contribute to the osmotic balance with the extracellular environment, enhance the stability of enzymes, and preserve the integrity of biological membranes (41). The composition of the osmolytes that are accumulated depends on the type of osmotic shock (i.e., ionic stress versus nonionic stress) and the presence of specific compatible solutes in the medium. In addition to their role as osmotic balancers, compatible solutes provide protection against high temperature, freeze-thaw treatment, and drying (41, 43).

When the osmotic pressure of the medium is increased, the tolerance of microorganisms to high hydrostatic pressure is enhanced. Baroprotective effects of sodium chloride or sugars have been observed in studies with Escherichia coli, Saccharomyces cerevisiae, Zygosaccharomyces spp., and Rhodoturula rubra (14, 17, 24, 25, 36). The influence of salts or sugars on the water activity (aw) of foods or suspension media does not explain the marked baroprotective effects of these solutes (21, 29), and it has been suggested that specific interactions between sugars and biological macromolecules contribute to their baroprotective effects (11). A comparable protective effect against pressure inactivation of Lactococcus lactis was achieved by addition of 3 M NaCl or 0.5 M sucrose, which resulted in aw values of 0.917 and 0.985, respectively (21). Furthermore, ionic and nonionic solutes had different effects on physiological properties of pressure-treated cells. Sucrose preserved the metabolic activity and membrane integrity of the cells during the high-pressure treatment, whereas salt preserved the membrane integrity but not the metabolic activity. L. lactis confronted with a decreased aw accumulates different compatible solutes depending on whether the reduction in the aw is achieved with an ionic solute or a nonionic solute (20), and preliminary evidence has suggested that the accumulation of compatible solutes is involved in the baroprotective effect of NaCl (21).

The inactivation of membrane-bound enzymes is a sensitive indicator of pressure-mediated sublethal injury of bacteria. The ATP-dependent multiple-drug-resistance (MDR) transporter HorA is inactivated prior to cell death in Lactobacillus plantarum during high-pressure treatment (38, 39), and the pressure-mediated inactivation of HorA depends on the membrane composition (39). In L. plantarum and L. lactis inactivation of the membrane-bound ATPases and reductions in the intracellular pH were observed after high-pressure treatment (22, 44).

Since membranes and membrane-bound enzymes are among the main targets of high-pressure inactivation, we directed our attention to the membrane-bound MDR transport enzymes of L. lactis. In L. lactis at least four drug extrusion activities have been detected (3, 20). One of these systems, LmrP, is a secondary drug transporter comprising 408 amino acid residues with 12 putative membrane-spanning segments (1-3, 40). The protein is homologous to several drug transporters belonging to the toxin-extruding antiporter group. This system utilizes the membrane potential and the transmembrane proton gradient mediating an electrogenic nH+-drug antiport reaction (n >= 2) to drive the extrusion of drugs out of the cell (4, 28).

The aim of this investigation was to obtain insight into mechanisms of baroprotection by NaCl and sucrose. Therefore, we determined the effects of sucrose and NaCl on the intracellular pH and membrane protein LmrP of L. lactis cells during and after high-pressure treatment. Based on previous investigations on the baroprotective effects of NaCl and sucrose (21), in this study the NaCl and sucrose levels were chosen to examine comparable baroprotective effects on cell counts and sublethal injury of L. lactis. Furthermore, the effects of solutes at the level of the membrane phase transition under normal and high-pressure conditions were studied.


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MATERIALS AND METHODS
 
Chemicals.
HEPES, 2-N-morpholineethanesulfonic acid (MES), valinomycin, and nigericin were obtained from Sigma-Aldrich (Steinheim, Germany), 5 (and 6-)-carboxyfluorescein diacetate N-succimidyl ester (cFDASE) was obtained from Fulka (Buchs, Switzerland), and 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) was obtained from Molecular Probes (Eugene, Oreg.). All other chemicals and M17 medium were analytical grade and were obtained from Merck (Darmstadt, Germany).

Milk buffer.
The milk buffer used contained the same amounts of minerals and lactose that whey from rennet casein contains; the buffer contained (per liter) 1.10 g of KCl, 0.71 g of MgSO4 · 7H2O, 1.87 g of Na2HPO4 · 2H2O, 1.00 g of CaSO4 · 2H2O, 0.99 g of CaCl2 · 2H2O, 2.00 g of citric acid, and 52.00 g of lactose. The pH was adjusted to 6.5, 6.0, 5.0, and 4.0 with KOH (1 M).

Bacterium and culture conditions.
L. lactis subsp. cremoris MG1363 was grown at 30°C in M17 broth supplemented with 1% glucose (GM17 broth).

Labeling of cells with 5 (and 6-)-carboxyfluorescein succimidyl ester for determination of the internal pH (pHin).
The fluorescence method developed by Breeuwer et al. (5) was adapted to L. lactis subsp. cremoris MG1363 (22). Cells from an overnight culture were washed and resuspended in 50 mM HEPES buffer (pH 8.0). Subsequently, the cells were incubated for 15 min at 30°C in the presence of 10.0 µM cFDASE, washed, and resuspended in 50 mM potassium phosphate buffer (pH 7.0). To eliminate nonconjugated 5 (and 6-)-carboxyfluorescein succimidyl ester, glucose (final concentration, 10 mM) was added, and the cells were incubated for an additional 30 min at 30°C. The cells were then washed twice, resuspended in the corresponding milk buffer, and placed on ice until they were used.

Pressure treatment of cell suspensions.
The cells of an overnight culture were harvested by centrifugation (15 min at 5,500 x g), washed, and resuspended in milk buffer or milk buffer with additives to a concentration of about 109 CFU · ml–1. The cells (2 ml) were suspended in sterile plastic micro test tubes, which were sealed with silicon stoppers and stored on ice until they were pressurized (the storage on ice was for up to 90 min). Samples were randomized to exclude a possible influence of the storage time at 0°C on the pressure sensitivity. The pressure chamber was heated and cooled to 20°C prior to pressurization with a thermostat jacket connected to a water bath. The compression and decompression rate was 200 MPa min–1, and the temperature increase due to compression was 6°C or less. Samples were energized with 10 mM glucose and placed in the pressure chamber 5 min prior to treatment to equilibrate the sample temperature. Cells were exposed to pressures of 200, 300, and 600 MPa for various times (0 to 120 min). Following the release of pressure the samples were stored on ice for determination of viable cell counts and further tests.

Enumeration of viable cells.
The cell suspensions from each vial were serially diluted with saline immediately after the pressure treatment and were surface plated on GM17 agar. The plates were incubated for 24 h at 30°C. The data presented below are means ± standard deviations obtained from two or three independent experiments.

In situ measurement of pHin.
Fluorescence was measured under hydrostatic pressures as described previously (22) in a pressure chamber equipped with a cylindrical sapphire window (10 by 8 mm). Two milliliters of stained cells was placed in the pressure chamber and energized with 10 mM glucose. The lid was closed and connected with an optical fiber to a spectrofluorometer (LS-50B luminescence spectrometer; Perkin-Elmer, Überlingen, Germany). Fluorescence intensities were measured at excitation wavelengths of 485 and 410 nm by rapidly alternating the monochromator between the two wavelengths. The emission wavelength was 520, and the excitation and emission slit widths were 15 nm. The incubation temperature was 20°C, and the apparatus was allowed to sit for 5 min prior to high-pressure treatment to equilibrate the temperature.

Calibration curves were determined by using buffers with pH values ranging from 4.0 to 8.0. Buffers were prepared from milk buffer at pH 4.0, 5.0, and 6.0 and from 50 mM HEPES buffer at pH 7.0 and 8.0. The pHin and external pH were equilibrated by addition of valinomycin (1 µM) and nigericin (1 µM), and the fluorescence was determined as described above. The fluorescence intensities were measured over a 5-min period at either the ambient pressure or 200 MPa, and the means were calculated for each buffer. A calibration curve was established for each culture that was stained and pressure treated on a single day. The pH values of buffers during pressure treatment were calculated as previously described (22).

Reversibility test.
After the pressure treatment, 1-ml portions of samples were placed in a cuvette, and the fluorescence intensities were determined as described above. After 3 min of equilibration, glucose was added to a final concentration of 10 mM, and the changes in pHin were monitored for up to 30 min.

LmrP activity assay.
The method developed by Ulmer et al. (38, 39) for HorA activity was used to determine the MDR transport activity of L. lactis. Extrusion of ethidium bromide in L. lactis MG1363 occurs predominantly with the LmrP transporter and is inhibited by ionophores that dissipate the proton motive force (1, 3, 4). We verified for the assay system described here that ethidium bromide efflux depended on the proton motive force, and therefore, MDR transport activity was reported as LmrP activity. After addition of the ionophores valinomycin and nigericin to dissipate the difference in pH ({Delta}pH) and the potassium membrane potential, the ethidium bromide efflux ceased (data not shown). One milliliter of a pressure-treated cell suspension was harvested by centrifugation and resuspended in 1 ml of phosphate buffer with 40 µM ethidium bromide. Samples were mixed and incubated at 30°C for 2 h in the dark. After incubation, the cells were harvested, resuspended in phosphate buffer, and transferred to black microtiter plates. The fluorescence of cell suspensions was measured after addition of glucose by using excitation and emission wavelengths of 485 and 595 nm, respectively. MDR transport-mediated ethidium bromide efflux results in decreased ethidium bromide fluorescence. The initial slopes of the curves were calculated with the Sigma Plot software and are reported below as LmrP activity.

Determination of the temperature-dependent phase state of the membrane by FT-IR spectroscopy.
Lipid phase transitions were measured by Fourier transform infrared (FT-IR) spectroscopy by using methods described previously (18, 39). The cells in 2 ml were harvested by centrifugation (3 min at 10,000 x g [relative centrifugal force]), washed twice, and incubated for 30 min in milk buffer, milk buffer with 0.5 M sucrose, or milk buffer with 4 M NaCl. After incubation, the cells were harvested, and the pellets were poured into a 20-µm-thick infrared cell with CaF2 windows. The FT-IR spectra were recorded with an Equinox 55 spectrometer (Bruker, Ettlingen, Germany) equipped with a liquid-nitrogen-cooled MCT (HgCdTe) detector. Twenty scans were averaged for each temperature point. Spectra were obtained every 2°C between 0 and 30°C and every 5°C between 30 and 40°C. The infrared cell was controlled by an external water thermostat.

Determination of the temperature- and pressure-dependent phase state of the membrane by fluorescence spectroscopy.
Laurdan fluorescence spectroscopy was used to detect phase changes in the lipid bilayer membrane. Laurdan (Molecular Probes) is an amphiphilic fluorescence probe which allows determination of gel-to-fluid phase transitions in biological membranes (26, 27, 39). The cells of an overnight culture were harvested by centrifugation, washed twice, and resuspended in milk buffer or milk buffer with additives. The cell suspensions were each diluted to an optical density at 590 nm of 1. A Laurdan stock solution in ethanol (2 mmol liter–1) was added to each cell suspension to obtain an effective staining concentration of 40 µmol liter–1. The cells were stained for 30 min at 30°C in the dark. After incubation the cells were washed twice, resuspended in the corresponding milk buffer, and placed on ice until they were used.

For determination of the temperature-dependent phase state of the membrane, stained cells were pipetted into a 2-ml cuvette (Sarstedt, Nümbrecht, Germany), and the Laurdan emission spectra were recorded with a spectrofluorometer (LS-50B luminescence spectrometer; Perkin-Elmer). The excitation wavelength was 360 nm, and the emission wavelength ranged from 380 to 550 nm. The generalized polarization (GP) was calculated as follows: GP = (I440nm – I490nm)/(I440nm + I490nm), where I440nm and I490nm are the relative fluorescence intensities at 440 and 490 nm, respectively (24). The temperature was controlled by a circulating water bath. Spectra were obtained every 10°C between 10 and 40°C. For the high-pressure fluorescence studies the pressure chamber described above, equipped with a cylindrical sapphire window (10 by 8 mm), was used. The pressure steps were 50-MPa steps with a ramp of 200 MPa min–1. The time allowed for equilibration after each pressure step was 3 min. The data presented below are means ± standard deviations obtained from two or three independent experiments.

Preparation of cell extracts.
To detect the accumulation of osmolytes in cells exposed to osmotic shock, cell extracts were prepared by the following method. Cells in the stationary phase (109 CFU · ml–1) were harvested by centrifugation (3 min at 9,000 x g) after incubation for 30 min in milk buffer or milk buffer with additives. The cell pellets were resuspended in sodium dodecyl sulfate buffer (100 mM Tris-HCl [pH 9.5], 1% [wt/vol] sodium dodecyl sulfate), and the cells were lysed by ultrasonication (three 1-min treatments; 30% cycle; 100% power; Bandelin Sonoplus). After centrifugation at 14,000 x g for 30 min at 4°C, the total supernatant fraction was recovered for analysis.

Determination of accumulated osmolytes by high-performance liquid chromatography analysis of cell extracts.
The method developed by Clarke et al. (8) was used for detection of sugars and amino acids in cell extracts of L. lactis. All experiments were performed with a high-pressure liquid chromatograph from Gynkotek. The system consisted of a model 480 gradient pump, a Gina 50 autosampler, a column thermostat, and an ED40 electrochemical detector containing a gold working electrode and an pH reference electrode. Norleucine was used as the internal standard.


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RESULTS
 
Effects of low-molecular-weight solutes during high-pressure treatment of L. lactis.
The protective effects of NaCl and sucrose were compared at pressures of 200, 300, and 600 MPa. At 200 MPa, a reduction in the cell count in milk buffer was observed after 40 min of treatment, and after 120 min the reduction was 2 logs (data not shown) (22). Addition of 0.5 M sucrose and 4 M NaCl protected the cells against inactivation, and the cell counts were essentially unaltered after 120 min of treatment (data not shown).

The inactivation curves for the bacteria at 300 MPa in milk buffer and milk buffer with 0.5 M sucrose had the typical sigmoid asymmetric shape (Fig. 1A). In milk buffer the inactivation phase started after 2 min of treatment, and after 20 min there was a reduction in the cell count of 5 logs. The results obtained with sucrose showed that this compound had a protective effect on the viability of L. lactis. The shoulder of the sigmoid asymmetric shape was extended until 8 min, and the inactivation phase was retarded. Addition of 4 M NaCl had a strong protective effect. Treatment for up to 60 min did not result in a significant reduction in cell counts, and treatment for 120 min reduced the cell count by 1 log.



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  		FIG. 1. Inactivation kinetics of L. lactis subsp. cremoris MG1363 in milk buffer (•), milk buffer with 0.5 M sucrose ({circ}), and milk buffer with 4 M NaCl ({blacktriangledown}) after pressure treatments at 300 MPa (A) and 600 MPa (B) and 20°C. The values were obtained by comparing the viable cell counts on GM17 medium with the viable cell counts of untreated cultures. The cell counts for control cultures were (2.4 ± 0.8) x 109 CFU ml–1. The values are means ± standard deviations for two independent experiments.

The inactivation curves at 600 MPa are shown in Fig. 1B. Treatment in milk buffer or milk buffer with 0.5 M sucrose resulted in rapid inactivation; after the buildup of pressure the cell counts were reduced by 4 to 5 logs. The strong protective effect of 4 M NaCl was apparent; even after treatment for 60 min the inactivation did not reach the same level as the levels in treatments with sucrose and without additives.

Effect of high pressure on the pHin of L. lactis.
The effect of treatment at 200 MPa on the pHin of L. lactis in milk buffer and milk buffer with additives is shown in Fig. 2. When the pHin of the energized cells at ambient pressure was used for comparison, the addition of solutes caused a decrease in the pHin, reducing the {Delta}pH of the cells (Fig. 2 and Table 1). An immediate decrease in the pHin occurred during compression, and the final value was reached 120 s after compression for treatments in milk buffer and milk buffer with sucrose. During treatment with 4 M NaCl, the final pHin was reached after 60 s, which corresponded to the time that it took for the pressure to build up. The final pHin for all treatments was about 5.2 and was correlated with the pH of the buffer at 200 MPa. The dissipation of {Delta}pH indicated that the regulation of pHin was impaired in these cells.



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  		FIG. 2. Change in the pHin of L. lactis subsp. cremoris MG1363 during treatment at 200 MPa and 20°C in milk buffer with additives. Samples were energized with 10 mM glucose and placed in the pressure chamber 5 min prior to treatment to equilibrate the sample temperature. (A) Milk buffer without additives; (B) milk buffer with 4 M NaCl; (C) milk buffer with 0.5 M sucrose. The data are the fluorescence data for cFDASE before and during pressure treatment. The pHin values calculated based on pressure treatments of depolarized cells are also indicated. The compression rate was 200 MPa min–1, and the ramp time was 60 s. The shaded areas indicate the ramp-up time required to compress the samples from 0.1 to 200 mPa.


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  		TABLE 1. {Delta}pH of untreated cells of L. lactis and of cells after pressure treatment for various times in milk buffer and in milk buffer with additives

Determination of the pHin of energized cells after pressure treatment indicated that a partial {Delta}pH was restored after 20 min of treatment in milk buffer (Table1). In agreement with previous results under comparable conditions (22), the buildup of pressure resulted in a reduction in the capacity to restore a {Delta}pH, and the cells were able to restore only 60% of the initial pH gradient. After treatments that lasted for 30 min, the cells were not able to restore the {Delta}pH. The loss of the capacity to restore a {Delta}pH was prevented by addition of sucrose to milk buffer. After 120 min of treatment in milk buffer with 0.5 M sucrose the cells were still able to restore a {Delta}pH of about 0.2 ± 0.1 pH unit. Addition of 4 M NaCl reduced the {Delta}pH of the untreated cells to 0.2 ± 0.1 pH unit. Pressure-treated cells were not able to increase the pHin within 30 min after decompression. Because these cells could be cultivated on M17 agar plates, they apparently recovered after longer incubation times in the presence of nutrients.

LmrP activity of pressurized cells.
According to the HorA assay of Ulmer et al. (38, 39), the effect of pressure on LmrP activity can be determined by comparing the ethidium bromide efflux of pressurized cells with the ethidium bromide efflux of starved and untreated cells. Figure 3 shows a comparison of the LmrP activities after treatments at 200 MPa in milk buffer and milk buffer with additives. Because LmrP is a proton motive force-dependent transport enzyme, the decreased LmrP activity of pressure-treated cells may result from (i) irreversible loss of the proton motive force or (ii) irreversible inactivation of the enzyme. No difference in LmrP activity was observed when untreated cells in milk buffer and untreated cells in milk buffer with sucrose were compared. High pressure impaired the activity of the enzyme. In milk buffer the LmrP activity was reduced after 8 min and reached the level in starved cells after 40 min. This effect correlated with the loss of capacity to restore a {Delta}pH and the first reduction in cell counts. When 0.5 M sucrose was added, the activity of LmrP was protected. The pressure inactivation of the enzyme was retarded, and the cells showed activity after 40 and 60 min of treatment; however, the cells lost the activity after 120 min. However, no reduction in cell counts was reported, and the cells were able to restore a partial {Delta}pH after 120 min of treatment, indicating that inactivation of LmrP rather than the dissipation of the proton motive force caused the loss of MDR transport activity. Untreated cells in milk buffer with 4 M NaCl showed strongly reduced LmrP activity. The residual activity was reduced during the buildup of pressure, and after 1 min of treatment the activity reached the level in starved cells. These results were consistent with the absence of a transmembrane {Delta}pH, but the viability of the cells was not affected.



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  		FIG. 3. Baroprotective effects of additives on LmrP during high-pressure treatment at 200 MPa and 20°C. The activity of LmrP in pressurized cells was compared to the activity in untreated cells in milk buffer (•), in milk buffer with 0.5 M sucrose ({circ}), and in milk buffer with 4 M NaCl ({blacktriangledown}). The data are means ± standard deviations for two independent experiments.

Effects of osmolytes on the membrane phase state at different temperatures, as measured by FT-IR spectroscopy.
Since one of the targets of high-pressure inactivation is the membrane, we determined the effects of osmolytes on the membrane phase state at different temperatures and different pressures. The melting temperature (Tm) of the phase transition from the gel phase to the liquid crystalline phase was determined by measuring the wave number of the symmetric CH2 absorbance peak at wave numbers near 2,850 cm–1 at various temperatures and then plotting the wave number of the absorbance maximum versus temperature (Fig. 4). The internal vibrational modes of the lipid acyl chains were assigned on the basis of studies of polymethylenes and polymethylene chain compounds (30, 34). In the region from 2,800 to 3,100 cm–1, there are infrared absorption bands due to symmetric and antisymmetric modes of the methylene chain at about 2,850 and 2,920 cm–1, respectively. The wave numbers of these bands are conformation sensitive and thus respond to temperature- and pressure-induced changes of the trans/gauche ratio in acyl chains. The vibration mode (antisymmetric stretch) of the terminal CH3 group occurs at about 2,960 cm–1. The FT-IR melting curves were fitted to the following sigmoid function:

where y0 and A are the lower and upper asymptotes, respectively, T is the temperature, and b is a shape coefficient. The Tm of the membranes was defined as the turning point of the melting curve. All of the liquid crystalline or gel phase regions are assumed to be temperature independent and are characterized by a nearly horizontal slope. This method has been used previously to determine the Tms of E. coli (18) and L. plantarum (39). Cells were grown overnight at 30°C in GM17 medium. L. lactis cells suspended in milk buffer had a Tm of 21.4°C. Addition of sucrose and NaCl to the buffer lowered the Tm of the phase transition. The sucrose decreased the Tm of the cells from 21.4°C to around 16.8°C, while NaCl lowered it to 16.6°C.



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  		FIG. 4. Vibrational frequencies for the CH2 symmetric stretch of membrane lipids of L. lactis cells in milk buffer ({circ}), in milk buffer with 0.5 M sucrose ({square}), and in milk buffer with 4 NaCl ({triangleup}) as a function of temperature. The data are representative of the data for two independent experiments. Every experiment was done by heating the cells from 0 to 40°C. The curves represent the best fit of the logistic function (r2 >= 0.98).

Effects of osmolytes on the membrane phase state at different temperatures and different pressures, as measured by Laurdan fluorescence spectroscopy.
The temperature- and pressure-dependent membrane phase behavior was determined by Laurdan fluorescence spectroscopy. The Laurdan fluorescence spectrum depends on changes in environmental polarity induced by membrane phase transitions. A membrane phase transition from the liquid crystalline phase to the gel phase decreases the polarity around the fluorophor in fluid membranes, resulting in increased GP values. The temperature-dependent membrane phase behavior of L. lactis determined by FT-IR spectroscopy was confirmed by Laurdan fluorescence spectroscopy. In the presence of sucrose or NaCl, the increase in GP values corresponding to the phase transition from the liquid crystalline phase to the gel phase was shifted to temperatures that were lower than the temperatures for cells suspended in milk buffer (data not shown). GP values of Laurdan-stained cells of L. lactis suspended in milk buffer or milk buffer with additives at pressures ranging from 50 to 300 MPa are shown in Fig. 5. For cells in milk buffer, an increase in GP values with increasing pressure was observed, and the cytoplasmic membranes were in the gel phase at a pressure of 150 MPa or greater. In the presence of sucrose, the pressure that induced the liquid crystalline phase-gel phase transition was shifted by 50 MPa, and the gel phase was induced by pressures of 200 MPa or greater. For cells suspended in milk buffer with NaCl the GP values did not increase with increasing pressure; at pressures up to 300 MPa their membranes stayed in the liquid crystalline phase.



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  		FIG. 5. GP values of L. lactis cells stained with Laurdan under pressure conditions. The treatments were performed in milk buffer (•), in milk buffer with 0.5 M sucrose ({circ}), and in milk buffer with 4 NaCl ({blacktriangledown}). The data are representative of the data for two to four independent experiments.

Intracellular accumulation of osmolytes by osmotic upshock.
Lactic acid bacteria accumulate different osmolytes if the cells have been stressed with sugars (nonionic stress) or with salt (ionic stress) (12, 13, 16, 20, 23). Lactic acid bacteria, including L. lactis MG1363, react to osmotic upshock with sugars by equilibration of the intracellular and extracellular sugar concentrations. Upon osmotic upshock with ionic solutes, compatible solutes, such as glycine betaine, are actively transported inside the cell. To verify the accumulation of osmolytes by L. lactis under conditions employed in our experiments, we determined qualitatively the main osmolytes in L. lactis cells after 30 min of incubation in milk buffer, milk buffer with 0.5 M sucrose, and milk buffer with 4 M NaCl. In agreement with previously published data, the cells exhibited sucrose uptake as a physiological response to the nonelectrolyte stress. Cells incubated with NaCl contained more than threefold more lactose than cells incubated in the absence of osmolytes contained. Furthermore, accumulation of an unknown osmolyte was apparent in the cells incubated in the presence of NaCl.


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DISCUSSION
 
In this study the effects of addition of NaCl and sugars on the survival and physiological activity of L. lactis during high-pressure treatment were evaluated. Furthermore, the effects of solutes on the pressure- and temperature-induced membrane phase transition were determined. Addition of 4 M NaCl or 0.5 M sucrose protected L. lactis against inactivation at 200 MPa. In milk buffer, the loss of viability after treatments correlated with reduced LmrP activity and the loss of the ability to restore a {Delta}pH after pressure treatment, in agreement with results obtained previously under comparable conditions (22). Addition of sucrose protected against pressure-induced inactivation of LmrP, extended the ability of the cells to restore a {Delta}pH, and shifted the Tm of the membrane to lower temperatures and higher pressures. Addition of NaCl at the ambient pressure dissipated the transmembrane {Delta}pH and strongly reduced the activity of the {Delta}pH-dependent MDR transporter LmrP. Nevertheless, NaCl protected L. lactis against the bactericidal effect of pressure. Comparison of the baroprotective effects of ionic and nonionic solutes suggests that the baroprotective effect is dependent on the response of microorganisms to the osmotic upshock, as well as specific interactions of the solutes with biomolecules.

Our work confirmed previous findings that membrane-bound enzymes are a major target for pressure-mediated sublethal injury and cell death. Pressure induces a phase transition of the bacterial membrane like a temperature downshift, and membrane proteins are reversibly inactivated when the membrane undergoes a phase transition to the gel phase (32). This reversible inactivation becomes irreversible after several minutes at a pressure of more than 200 MPa (15). When bacterial cells are subjected to pressure treatment, membrane-bound enzymes, such as HorA (38, 39), LmrP (this study), and enzymes involved in pH homeostasis (22, 44), are irreversibly inactivated prior to cell death. Therefore, pressure treatment of bacteria may result in sublethally injured cells which recover when they are transferred to nonselective media after pressure treatment because of de novo protein biosynthesis or refolding of denatured integral membrane proteins. However, sublethally injured cells are rapidly killed in the presence of physical or chemical stresses after pressure treatment.

Lipids in biological membranes are in the fluid (liquid crystalline) phase, which allows fast lateral movement of molecules. The removal of water, a pressure upshift, or a temperature downshift in one-component phospoholipid bilayers induces a sharp phase transition from the liquid crystalline phase to a gel phase (42). A membrane phase transition to the gel phase per se is not lethal to bacteria, but previous investigations suggested that the fluidity of the bacterial membrane has a direct role in the pressure resistance of bacteria (7). Analogous to the response of piezophilic bacteria to elevated growth pressure (1), increased pressure resistance of bacteria with a more fluid membrane was expected. Indeed, exponentially growing cells of E. coli were more resistant to pressure when the membrane was more fluid (7). Likewise, we found in this study that addition of NaCl and sucrose maintained the bacterial membrane in a more fluid state during pressure treatment and enhanced the resistance of bacteria to pressure. In contrast, it was observed previously that stationary cells of E. coli and L. plantarum were more resistant to pressure when the cells had a more rigid membrane than when the cells had a more fluid membrane (7, 39). Furthermore, it is well established that bacteria exhibit a temperature optimum for pressure resistance, which is a few degrees below the cultivation temperature. Increasing and decreasing the pressurization temperature from the temperature optimum for baroresistance result in increased and decreased membrane fluidity during pressurization, respectively, but in both cases increased sensitivity to pressure is observed (35). Therefore, it is currently not possible to establish a clear correlation between the membrane fluidity of bacteria and the resistance of the bacteria to pressure. It is more likely that the membrane fluidity has an indirect effect on the pressure resistance of bacteria because the irreversible denaturation of membrane-bound proteins by pressure is strongly dependent on the properties of the cytoplasmic membrane (15, 39). However, pressure-temperature phase diagrams of integral membrane proteins in phosphilipid bilayers that could provide experimental evidence for this hypothesis are currently not available.

Addition of disaccharides to biological systems protects against changes in the physical state of membrane lipids and maintains the native structure of proteins during physical and chemical stresses (9, 10, 18, 37, 45). Timasheff et al. (37) have suggested that stabilization of proteins in sugar solutions is due to the preferential exclusion of the sugar from contact with the protein surface. The native protein structure is stabilized because denaturation of the protein molecule leads to a larger contact surface between the protein and the solvent and therefore enhances this thermodynamically unfavorable effect. The protective effect of sugars on biomolecules is employed by organisms in all kingdoms as a reaction to chemical stress (e.g., drying [10]) or to physical stresses, such as heat (33). Piezophilic bacteria accumulate osmolytes in response to elevated growth pressure (19).

High concentrations of sugars (lactose and sucrose) result in only transient osmotic stress in L. lactis because the cells are able to equilibrate the extra- and intracellular concentrations of sucrose and lactose (13), and we confirmed the accumulation of sucrose in response to sucrose-mediated osmotic upshock in our assay system. Thus, in our experiments with milk buffer and sucrose, high levels of sucrose were present both intracellularly and extracellularly. In accordance with the protective effects of disaccharides on membranes, addition of sucrose reduced the Tm of the cell membrane and delayed the liquid crystalline phase-gel phase transition during high-pressure treatment. Furthermore, inactivation of the MDR transport enzyme LmrP, as well as inactivation of membrane-bound enzymes involved in pH homeostasis, was delayed or prevented, suggesting that there was stabilization of the protein structure in the presence of sucrose.

High salt concentrations are detrimental to mesophilic microorganisms. In nonhalophilic bacteria, the most efficient strategy to cope with hyperosmotic conditions is cytoplasmic accumulation of compatibles solutes. These solutes can be accumulated up to high concentrations without inhibiting enzyme function and in addition are able to stabilize proteins (31). The compatible solutes not only confer increased tolerance towards higher osmolarity but also confer increased tolerance towards desiccation, freezing, and elevated temperatures. The process responsible for stabilization is called preferential exclusion or preferential hydration. In the presence of NaCl, lactic acid bacteria preferentially accumulate glycine betaine as a compatible solute. If glycine betaine is not available in the medium, amino acids or disaccharides are accumulated in response to the osmotic upshock (12, 13); L. lactis in our experimental setup accumulated lactose and unknown osmolytes in the presence of 4 M NaCl. Thus, in our experiments with milk buffer and NaCl, high levels of lactose were present intracellularly, yet no protective disaccharides or compatible solutes were present extracellularly. This condition was lethal to L. lactis even at the ambient pressure after exposure for several hours (data not shown). At the ambient pressure, the {Delta}pH was almost fully dissipated by addition of 4 M NaCl, and LmrP activity was reduced to 30% of the activity in untreated cells.

Remarkably, the combination of lethal NaCl levels and lethal pressure provided protection against cell death. Furthermore, addition of NaCl reduced the Tm of the cell membrane and increased the pressure required to induce a phase transition from the liquid crystalline phase to the gel phase. Monovalent cations do not affect the phase properties of phospholipid bilayers (6). Therefore, the protective effect of NaCl on the cell membrane of L. lactis must be attributed to the presence of compatible solutes accumulated intracellularly. In contrast to pressure treatments with sucrose addition, in the presence of NaCl only intracellular components of the cells benefit from the protective effect of compatible solutes. Because compatible solutes are present only in the cytoplasm, the outer leaflet of the membrane and cell wall-bound enzymes remain unprotected, and, compared to the concentrations of sucrose, higher salt concentrations are required to achieve an equivalent protective effect.

In conclusion, accumulation of disaccharides or compatible solutes protects vegetative bacteria against pressure-mediated cell death. Piezophilic deep-sea bacteria accumulate osmolytes in response to elevated hydrostatic pressure within the growth range of the organisms (19). Pressure-dependent accumulation of osmolytes has not been described for piezosensitive bacteria; however, protective osmolytes are accumulated in response to osmotic upshock. Ionic solutes provide only partial or asymmetric protection as only intracellular components of the cell are protected. Therefore, baroprotection with ionic solutes requires higher concentrations of the osmolytes than baroprotection by disaccharides requires.


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ACKNOWLEDGMENTS
 
We thank Alexander Schober, Bernhard Balg, Sabine Eichl, and Dominika Barác for their contributions to this work.

We are grateful for financial support provided by grant FOR 358/1 from the Deutsche Forschungsgemeinschaft.


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FOOTNOTES
 
* Corresponding author. Mailing address: TU München, Lehrstuhl Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany. Phone: 49 8161 713204. Fax: 49 8161 713327. E-mail: michael.gaenzle{at}wzw.tum.de. Back


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Applied and Environmental Microbiology, April 2004, p. 2013-2020, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2013-2020.2004
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