Glutathione Protects Lactococcus lactis against Acid Stress

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2007, p. 5268–5275 0099-2240/07/$08.00⫹0 doi:10.1128/AEM.02787-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 16

Glutathione Protects Lactococcus lactis against Acid Stress䌤 Juan Zhang,1 Rui-Yan Fu,1,4 Jeroen Hugenholtz,3 Yin Li,2* and Jian Chen1* Key Laboratory of Industrial Biotechnology, Ministry of Education, and School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China1; Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China2; Wageningen Centre for Food Sciences, Kluyver Centre for Genomics of Industrial Fermentation, and NIZO food research, 6710 BA Ede, The Netherlands3; and Laboratory of Tea Biochemistry & Biotechnology, Ministry of Agriculture and Ministry of Education, Anhui Agricultural University, Hefei 230036, People’s Republic of China4 Received 29 November 2006/Accepted 19 June 2007

Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739–5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH. Lactococcus lactis is a neutrophilic bacterium whose optimal growth occurs within an extracellular pH range of 6.3 to 6.9 (9). Growth of L. lactis is typified by the generation of acidic end products (mainly lactic acid), which results in medium acidification and subsequent acid stress. Acid stress has detrimental effects on the cellular physiology of L. lactis, including damage to the cell membrane and inhibition of enzymes and transport systems (14). Low pH is therefore considered a growth-limiting factor for L. lactis grown in milk or weakly buffered media. Consequently, the capability of L. lactis to survive, grow, and metabolize actively at a low pH will greatly influence its industrial performance as a starter. A number of acid stress resistance mechanisms in L. lactis have been identified and characterized. The primary mechanism of L. lactis for surviving low pH is to control the intracellular pH (pHi) by membrane-bound FoF1 ATPase, which translocates protons to the environment at the expense of ATP (9, 21). Other mechanisms include generation of alkaline substances by amino acid catabolism (e.g., deamination) (6, 24). L. lactis also develops a complex adaptive response to acid stress which is dependent on the synthesis of proteins such as heat shock proteins and proteinases (9). Although the native acid stress resistance mechanisms in L. lactis were extensively studied, improving the acid stress resistance of L. lactis by introducing a xenobiotic compound whose metabolism is not directly related to acid stress resistance has not been investigated.

Glutathione (␥-Glu-Cys-Gly) (GSH) is the major nonprotein thiol compound in living cells. The major physiological role of GSH in living organisms is to maintain a redox balance (4). However, recent studies showed that GSH is also involved in bacterial acid stress resistance (23), osmotic-stress resistance (28), chlorine compound defense (5), and toxic electrophile detoxification (10). Most of these new physiological roles of GSH were found in gram-negative bacteria, such as Escherichia coli and Rhizobium tropici. To date, little is known about the physiological role of GSH in gram-positive bacteria. We pioneered the research investigating the physiological roles of GSH in lactic acid bacteria, using L. lactis as a model organism. In previous studies, we have shown that the GSH imported by L. lactis subsp. cremoris SK11 protects the host against H2O2induced oxidative stress (17). Subsequently we constructed a metabolically engineered L. lactis subsp. cremoris strain, NZ9000(pNZ3203), which successfully produces GSH (18), and in a follow-up study we showed that the intracellularly produced GSH in strain NZ9000(pNZ3203) protects the host against high-dose oxidative-stress treatment (150 mM H2O2 for 15 min and 30 ␮M menadione for 40 min) (11). These studies demonstrated that the GSH, either imported or produced by L. lactis cells, is physiologically beneficial to the host in terms of increasing the oxidative-stress resistance. The aim of the present study was to investigate if GSH can protect L. lactis against acid stress. The model organisms used in this study were L. lactis SK11 and L. lactis NZ9000. L. lactis SK11 cannot synthesize GSH but can import GSH from the medium (17). L. lactis NZ9000 can neither synthesize nor import GSH on its own but can produce GSH upon introduction of a plasmid, pNZ3203, with the E. coli gshA and gshB genes (18). Here we present evidence that L. lactis cells containing GSH have a significantly increased survival rate when challenged at pH 4.0, a pH where most of the growth arrest of L. lactis occurs (9). The protective role of GSH in L. lactis against

* Corresponding author. Mailing address for Jian Chen: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, People’s Republic of China. Phone and fax: 86-510-85918309. E-mail: [email protected]. Mailing address for Yin Li: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, People’s Republic of China. Phone and fax: 86-10-64807485. E-mail: [email protected]. 䌤 Published ahead of print on 29 June 2007. 5268

GLUTATHIONE PROTECTS L. LACTIS AGAINST ACID STRESS

VOL. 73, 2007 TABLE 1. Strains and plasmids used in this study Strain or plasmid

Relevant characteristics

Strains L. lactis subsp. cremoris SK11 L. lactis subsp. cremoris NZ9000 Plasmids pNZ8148 pNZ3203

a

a

Reference

Can import GSH from medium but cannot synthesize GSH Strain MG1363 pepN::nisRK, which can neither import nor synthesize GSH

17

Cmr, inducible expression vector carrying nisA promoter Cmr, pNZ8148 derivative containing functional E. coli gshA and gshB genes

19

18

18

Cmr, chloramphenicol resistance.

acid stress is of interest to dairy industries, e.g., to maintain a higher viability of cells during a progressive decrease of pH. MATERIALS AND METHODS Chemicals. M17 broth was purchased from Oxoid (Basingstoke, Hampshire, United Kingdom). GSH, GSH reductase, NADPH, NAD⫹, NADP⫹, glyceraldehyde-3-phosphate, fructose l,6-diphosphate, phosphoenolpyruvate, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, 5,5⬘-dithiobis (2-nitrobenzoic acid), valinomycin, nigericin, and HEPES were purchased from Sigma-Aldrich (Steinheim, Germany). 5 (and 6-)-Carboxyfluorescein diacetate N-succinimidyl ester was purchased from Axxora Life Science, Inc. (San Diego, CA). All inorganic compounds were of reagent grade or higher quality. Bacterial strains and culture conditions. Strains and plasmids used in this study are listed in Table 1. For strain SK11, inoculum was transferred from ⫺70°C frozen stock to chemically defined medium (CDM) supplemented with 5 g/liter lactose, incubated at 30°C statically for 16 h as a preculture. The preculture was then inoculated with an inoculum size of 1% (vol/vol) into CDM containing 3.2 mM GSH or not containing GSH to obtain SK11 cells with or without intracellular GSH. The detailed composition of the CDM was described in a previous study (17). For strain NZ9000(pNZ3203) and its control strain, NZ9000(pNZ8148), inocula were transferred from ⫺70°C frozen stocks to M17 broth supplemented with 5 g/liter glucose, incubated at 30°C statically for 16 h as precultures. The precultures were then inoculated with an inoculum size of 1% (vol/vol) into M17 broth supplemented with 5 mM L-cysteine. Chloramphenicol (5 ␮g/ml) was used as a selection marker for plasmids pNZ8148 and pNZ3203 in the preculture and the culture. Nisin (2 ng/ml) was added to the cultures of strain NZ9000(pNZ3203) and NZ9000(pNZ8148) at 2 h to induce the expression of genes gshA and gshB on pNZ3203. These two genes encode the ␥-glutamylcysteine synthetase and glutathione synthetase involved in GSH biosynthesis. The reasons that CDM was used for strain SK11 while M17 broth was used for strains NZ9000(pNZ3203) and NZ9000(pNZ8148) are as follows. First, strain SK11 can import the GSH present in M17 broth, while strain NZ9000 cannot (17). Therefore, a CDM is desirable for strain SK11 to obtain SK11 cells with or without intracellular GSH. Second, in a previous study, M17 broth supplemented with 5 mM L-cysteine was shown to be the best medium for NZ9000(pNZ3203) to produce GSH (18). Preparation of cell extracts. Bacteria were harvested by centrifugation. The cell pellets were washed twice with ice-cold saline (0.85% NaCl [wt/vol]) and resuspended in an equal volume of phosphate buffer B (0.2 M potassium phosphate, 2 mM EDTA; pH 7.0). Three milliliters of cell suspension was disrupted ultrasonically at 4°C for 40 cycles of 5 s (ACX 400 sonicator at 20 kHz; Sonic and Materials, Newton, MA). Cell debris was removed by centrifugation (10,000 ⫻ g for 10 min at 4°C), and the cell extracts were used for the GSH assay and the enzyme assay. GSH assay. Total GSH (reduced form, GSH, plus oxidized form, GSSG) was determined by the enzymatic recycling procedure as modified from the procedures of Tietze (29), which has been detailed in a previous study (17). Protein concentrations were determined by using the Lowry method with bovine serum albumin as a standard. Acid challenge. Portions (10 ml) of the cultures of L. lactis SK11 and L. lactis NZ9000 and its derivatives grown to different growth phases were centrifuged at

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10,000 ⫻ g for 5 min. Cell pellets were washed with saline to remove the residual medium. Cells were pelleted again and resuspended in 10 ml 100 mM potassium phosphate buffer adjusted to different pHs with lactic acid unless otherwise specified. After challenge for various times, a 1-ml portion was withdrawn, centrifuged at 20,000 ⫻ g for 1 min, and washed with saline to remove the residual acidified potassium phosphate buffer. Cells were resuspended in 1 ml saline and spread onto M17 plates at different dilutions to determine the survival rate. Measurement of pHi. pHi was measured by the fluorescence method developed by Breeuwer et al. (3) using 5 (and 6-)-carboxyfluorescein succimidyl ester as the fluorescent probe. Calibration curves establishing the relationship between extracellular pH and pHi for strains SK11(GSH⫹), SK11(GSH⫺), NZ9000 (pNZ3203), and NZ9000(pNZ8148) were, respectively, established to exclude artifacts caused by environmental conditions and different types of cells. Loading of cells with 5 (and 6-)-carboxyfluorescein succimidyl ester, determination of pHi, and calibration of pHi all followed the procedure described previously (3). The calibration of SK11(GSH⫹) and SK11(GSH⫺) cells yielded highly similar calibration curves (nearly overlapped). The calibration of NZ9000(pNZ3203) and NZ9000(pNZ8148) also yielded similar calibration curves (not overlapped). We therefore used individual calibration curves to calculate the pHi for NZ9000 (pNZ3203) and NZ9000(pNZ8148). Using cells of NZ9000(pNZ3203) as a model, we demonstrated that the use of different weak acids (lactic acid and acetic acid) does not significantly affect the calibration curve. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assay. Two hundred fifty microliters of 500 mM triethanolamine chloride (pH adjusted to 7.2 using 10 M NaOH), 100 ␮l of 50 mM Na arsenate, 100 ␮l of 50 mM cysteine-HCl, 100 ␮l of 10 mM NAD⫹, 400 ␮l of MilliQ water, and 40 ␮l of diluted cell extracts were added to the cuvette. Glyceraldehyde-3-phosphate (6.7 ␮l; 50 mg/ml) was added to the cuvette to start the reaction. The increase of absorbance at 340 nm was dynamically monitored at 30°C using a spectrophotometer (Biospec-1601; Shimadu Co., Kyoto, Japan). GAPDH activity was expressed as units per milligram of protein, where 1 U is defined as the amount of enzyme producing 1 ␮mol of NADH per min. PK assay. Pyruvate kinase (PK) activity was assayed using a previously described protocol (1) with modifications. Cell extracts were added to a solution containing 0.1 mM fructose l,6-diphosphate, 0.1 mM phosphoenolpyruvate, 0.1 mM ADP, 2 mM NADH, and 4 U of lactate dehydrogenase. Activity was determined from the rate of decrease in absorbance at 340 nm, with 1 U corresponding to the oxidation of 1 ␮mol of NADH per min at 37°C. HK assay. Hexose kinase (HK) activity was determined by amending the method of Barnard (2). The 3-ml reaction mixture contained the following: 1.1 ml of 0.1 M triethanolamine buffer (pH 7.6), 1.2 ml of 10% (wt/vol) ␤-D-glucose, 0.2 ml of 0.1 M MgCl2, 0.1 ml of 0.08 M ATP, 0.2 ml of 0.013 M NADP⫹, 0.1 ml of 15-U/ml glucose-6-phosphate dehydrogenase, and 0.1 ml of cell extracts. Activity was determined based on the rate of increase in absorbance at 340 nm, with 1 U corresponding to the formation of 1 ␮mol of catalyzed product per min at 37°C. Determination of intracellular ATP concentration. ATP was determined based on a previously described method (25) with modifications. The cellular metabolism was quenched by putting 30 ml cell suspension into liquid nitrogen for 3 min, to which 10 ml of 0.6 M HClO4 was added, and the mixture was stirred for 10 min. The supernatant was collected after centrifugation. The pellet was resuspended in 10 ml of 0.6 M HClO4 and stirred for 10 min and the supernatant collected. Both supernatants were blended and diluted to a final volume of 25 ml with 0.6 M HClO4. The pH of the mixture was adjusted 7.0 with 0.8 M KOH. The mixture was then filtrated to remove the crystal of KClO4. The intracellular ATP concentration was detected by Agilent 1100 high-performance liquid chromatography (Agilent Technologies, Palo Alto, CA) using a stable C18 column (Thermo Electron Corporation) under the following conditions: mobile phase, 80% of 10 mM potassium phosphate buffer (pH 7.0) and 20% of methanol; flow velocity, 1.2 ml/min; detector, 260 nm; column temperature, 25°C; injection volume, 10 ␮l; elution time, 10 min. Statistical analysis. Student’s t test was employed to investigate statistical differences. Samples with P values of ⬍0.05 were considered statistically different.

RESULTS GSH imported by L. lactis SK11 has protective role against acid stress. To address the question of whether the GSH imported by strain SK11 can protect the host against acid stress, cells of strain SK11 were grown in CDM supplemented

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FIG. 1. Effect of acid stress on the survival of L. lactis SK11(GSH⫹) cells and SK11(GSH⫺) cells. SK11(GSH⫹) cells were obtained by growing SK11 cells in CDM supplemented with 3.2 mM GSH. Cells were harvested at mid-exponential phase (6 h), mid-stationary phase (10 h), and later stationary phase (19 h). Cells were washed and resuspended in 100 mM potassium phosphate buffer adjusted to pH 4.0 with lactic acid and held at room temperature for 10 h. (A) Survival rate of SK11 cells. (B) Intracellular GSH content of SK11 cells before acid challenge. Error bars indicate standard deviations (n ⫽ 3). Statistically significant differences (P ⬍ 0.05) were determined by Student’s t test and are indicated with an asterisk.

with 3.2 mM GSH [designated SK11(GSH⫹) cells] or without GSH [designated SK11(GSH⫺) cells]. Cells grown to midexponential (6 h) phase, mid-stationary (10 h) phase, and later stationary (19 h) phase were withdrawn, and their intracellular GSH content and acid stress resistance were determined. Considering that lactic acid is the major acidic product during the growth of L. lactis, cells were challenged at pH 4.0 for 10 h. As shown in Fig. 1, the acid stress resistance of SK11 cells at mid-exponential phase was significantly lower than that of the cells grown to mid-stationary phase and later stationary phase, which is similar to the previously reported pattern of acid stress resistance of L. lactis MG1363 (8). The survival rates of SK11 (GSH⫹) cells harvested from all growth phases were significantly higher than those of SK11(GSH⫺) cells, suggesting a protective role of GSH against acid stress in SK11(GSH⫹) cells. The survival rates of SK11(GSH⫹) cells harvested from mid-exponential phase and mid-stationary phase were 7.9- and 8.1-fold those of SK11(GSH⫺) cells (Fig. 1A). This indicates that the protective effect of GSH did not increase over the

APPL. ENVIRON. MICROBIOL.

transition period from mid-exponential phase to mid-stationary phase. However, the survival rate of SK11(GSH⫹) cells harvested from later stationary phase was 15.9-fold that of SK11(GSH⫺) cells, suggesting a more-prominent protective effect of GSH when cells are getting old. The intracellular GSH content of SK11(GSH⫹) cells increased along with the increase of incubation time (Fig. 1B), which showed a positive, although not proportional, correlation with the increased survival rate of SK11(GSH⫹) cells (Fig. 1A). The above data demonstrated that the survival of SK11 (GSH⫹) cells is significantly higher than that of the control when cells encountered a mild acid challenge at pH 4.0, a pH where most of the growth arrest of L. lactis occurs (9). We therefore intended to investigate if GSH can protect SK11 cells against a severe acid challenge. Since the protective role of GSH was prominent in SK11(GSH⫹) cells harvested from later stationary phase, L. lactis SK11 incubated for 19 h (laterstationary-phase cells) was used in the following study. SK11 (GSH⫹) and SK11(GSH⫺) cells were challenged at pH 2.5. A 17.2-fold-increased survival of SK11(GSH⫹) cells, compared to that of SK11(GSH⫺) cells, was exclusively but reproducibly observed when cells were challenged at pH 2.5 for 30 min (Fig. 2A). No significant difference between the survival rates of the two cell types was observed when the challenge at pH 2.5 lasted less than or longer than 30 min. This profile is completely different from that with pH 4.0, where significant differences between the survival rates of the two cell types were observed after challenge for 4 h, and the difference then became larger (Fig. 2B). These data suggest that the presence of GSH in SK11(GSH⫹) cells can protect the host against a long-term mild acid challenge and a short-term severe acid challenge with lactic acid. We also tested the responses of SK11(GSH⫹) and SK11(GSH⫺) cells with pH 4.0 and pH 2.5 adjusted by acetic acid, and a similar protective role of GSH for strain SK11 (GSH⫹) was observed (data not shown). This suggests that the protection of GSH against acid stress is independent of the weak acid used. GSH produced by engineered cells of L. lactis NZ9000 also plays a protective role against acid stress. L. lactis NZ9000 is the host for the nisin-induced expression system (19). While strain SK11 can import GSH from the medium, strain NZ9000 can neither synthesize nor import GSH (17). In a previous study, we introduced the GSH biosynthetic capability into strain NZ9000 by transforming a pNZ8148 derivative, pNZ3203, in which the genes gshA and gshB from Escherichia coli encoding ␥-glutamyl cysteine synthetase and glutathione synthetase were cloned (18). Recently we demonstrated that the GSH produced by strain NZ9000(pNZ3203) can protect the host against the oxidative stress induced by H2O2 and menadione, presenting the first example of using a metabolic engineering approach to improve lactococcal stress resistance (11). To explore if the GSH produced by engineered cells of strain NZ9000 can protect the host against acid stress, we determined the acid stress resistance of strain NZ9000(pNZ3203) when challenged at pH 2.5 or pH 4.0 adjusted by lactic acid. Laterstationary-phase (incubation for 19 h) cells of strain NZ9000 (pNZ3203) and strain NZ9000(pNZ8148) were used. We observed that GSH plays a protective role for strain NZ9000 (pNZ3203), and the protective profile of GSH for strain

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FIG. 2. Effects of GSH on the survival of L. lactis SK11 and NZ9000 challenged at different pHs. For strain SK11, initial cell concentrations of SK11(GSH⫹) and SK11(GSH⫺) before acid challenge were 2.5 ⫻ 108 CFU/ml and 3.2 ⫻ 108 CFU/ml, respectively. The initial concentration of intracellular GSH in SK11(GSH⫹) was 29.8 nmol/mg of protein. For strain NZ9000, the initial cell concentrations of NZ9000(pNZ3203) and NZ9000(pNZ8148) were 2.0 ⫻ 109 CFU/ml and 2.3 ⫻ 109 CFU/ml, respectively. The initial concentration of intracellular GSH in NZ9000 (pNZ3203) was 26.8 nmol/mg of protein. Panels A and B represent the survival of L. lactis SK11(GSH⫹) cells and SK11(GSH⫺) cells at different pHs adjusted by lactic acid. Panels C and D represent the survival of L. lactis NZ9000(pNZ3203) and NZ9000(pNZ8148) at different pHs adjusted by lactic acid. Error bars indicate standard deviations (n ⫽ 3). Statistically significant differences (P ⬍ 0.05) were determined by Student’s t test and are indicated with an asterisk.

NZ9000(pNZ3203) was very similar to that for strain SK11 (GSH⫹): a short-term protective effect of a 15-fold-higher survival rate of strain NZ9000(pNZ3203) when challenged at pH 2.5 for 30 min (Fig. 2C) and a long-term protective effect of an 18-fold-higher survival rate of strain NZ9000(pNZ3203) when challenged at pH 4.0 for 10 h (Fig. 2D). A similar protective effect was observed when strains NZ9000(pNZ3203) and NZ9000(pNZ8148) were challenged with acid stress generated by acetic acid (data not shown). These observations lead to the conclusion that GSH, either imported or produced by L. lactis cells, can protect the host against acid stress. Lactococcal cells with intracellular GSH display a slow decline of intracellular pH upon acid challenge. We employed biochemical approaches to explore the mechanism of the protective role of GSH in acid stress resistance, starting with determining the changes of pHi of acid-stressed cells of strain SK11 and strain NZ9000. As shown in Fig. 3, SK11(GSH⫹) cells and NZ9000(pNZ3203) cells seem to have the capability of maintaining a significantly higher pHi and a slow decline of

pHi compared to that of the control. This suggests that the presence of GSH in both strains may effectively prevent the rapid decline of pHi with a short-term severe acid challenge and a long-term mild acid challenge. A detailed analysis of Fig. 3 revealed that the changes of pHi in strains SK11 and NZ9000 were different. First, the pHis of NZ9000(pNZ3203) cells and NZ9000(pNZ8148) cells were 0.60 ⫾ 0.05 higher than those of SK11(GSH⫹) cells and SK11(GSH⫺) cells (Fig. 3A and B), suggesting the pHi of NZ9000 cells is characteristically higher than that of SK11 cells. Second, the ⌬pHi between cells of NZ9000(pNZ3203) and NZ9000(pNZ8148) when challenged at pH 4.0 was nearly constant (0.18 at 2 h, 0.15 at 4 h, and 0.18 at 6 h). However, the ⌬pHi between cells of SK11(GSH⫹) and SK11(GSH⫺) when challenged at pH 4.0 increased continuously (0.09 at 2 h, 0.15 at 4 h, and 0.17 at 6 h) (Fig. 3A and B). This suggests that the significant decrease of pHi in NZ9000 (pNZ8148) during acid challenge happened primarily in the beginning, while the decrease of pHi in SK11(GSH⫺) was a gradual process.

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FIG. 3. Changes in the pHis of L. lactis SK11 and NZ9000 cells upon acid challenge. Later-stationary-phase (19 h) cells of SK11(GSH⫹), SK11 (GSH⫺), NZ9000(pNZ3203), and NZ9000(pNZ8148) were withdrawn and challenged at pH 2.5 or pH 4.0 adjusted by lactic acid or acetic acid for various times. Cells were pelleted and washed with saline to prepare for pHi determination. (A) pHis of SK11(GSH⫹) or SK11(GSH⫺) cells treated with lactic acid. (B) pHis of NZ9000(pNZ3203) or NZ9000(pNZ8148) cells treated with lactic acid. Error bars indicate standard deviations (n ⫽ 3). Statistically significant differences (P ⬍ 0.05) were determined by Student’s t test and are indicated with an asterisk.

Change in GSH level of L. lactis SK11 is different from that for L. lactis NZ9000 during acid challenge. The second approach we used to understand the protective role of GSH in acid stress resistance was to examine the changes in the intracellular GSH contents (GSHin) in SK11(GSH⫹) cells and NZ9000(pNZ3203) cells during acid challenge. A control experiment resuspending SK11(GSH⫹) cells in 100 mM potassium phosphate buffer (pH 7.0) for 10 h showed an 11.6% decrease in the GSHin. However, challenging the SK11 (GSH⫹) cells with 100 mM potassium phosphate buffer adjusted to pH 4.0 for 10 h resulted in a 92.4% decrease in the GSHin (Fig. 4A), suggesting the intracellular GSH was depleted during acid challenge. Further analysis revealed that a 6.3-fold decrease in the GSHin happened during the first 4 h of acid challenge, which corresponded to a 3.8-fold decrease in the survival of SK11(GSH⫹) cells. However, during the last 6 h

of acid challenge (from 4 h to 10 h), the GSHin further decreased 2.1-fold, while the survival of SK11(GSH⫹) cells strikingly decreased, by 25-fold. The steep decline of GSHin and mild mortality of SK11(GSH⫹) cells in the beginning might be an interesting clue for further understanding the role of GSH against acid stress in SK11. On the contrary, challenging the NZ9000(pNZ3203) cells at pH 4.0 for 10 h resulted in a 23.4% decrease in the GSHin (Fig. 4B), while the decrease in the GSHin was 14.3% when NZ9000(pNZ3203) cells were resuspended in pH 7.0 for 10 h (control; data not shown). To exclude the possibility that the slow decrease of GSHin in NZ9000(pNZ3203) was due to continuous biosynthesis, we determined the intracellular ATP concentration, since the availability of ATP determines the biosynthesis of GSH. We found the intracellular ATP in both NZ9000(pNZ3203) and NZ9000(pNZ8148) cells was depleted after acid stressing for

FIG. 4. Relationship between the intracellular GSH levels and the survival of acid-stressed SK11(GSH⫹) cells and NZ9000(pNZ3203) cells. Later-stationary-phase (19 h) cells were harvested, washed with saline, and resuspended at a concentration of 108 cells per ml. After challenge for various times, a sample was withdrawn, washed, resuspended, and divided into two portions. One portion was used for GSH determination, and the other one was used for viable cell counts. (A) SK11(GSH⫹) cells. (B) NZ9000(pNZ3203) cells. Error bars indicate standard deviations (n ⫽ 3).


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FIG. 5. GAPDH and PK activities of strains SK11 and NZ9000 upon acid challenge. Later-stationary-phase (19 h) cells of SK11(GSH⫹), SK11 (GSH⫺), NZ9000(pNZ3203), or NZ9000(pNZ8148) were used. (A) GAPDH activities of SK11(GSH⫹) or SK11(GSH⫺) cells. (B) GAPDH activities of NZ9000(pNZ3203) or NZ9000(pNZ8148) cells. Error bars indicate standard deviations (n ⫽ 3). Statistically significant differences (P ⬍ 0.05) were determined by Student’s t test and are indicated with an asterisk.

6 h, and the intracellular ATP concentrations in the two cell types decreased at comparable rates (data not shown). This indirect evidence suggests that GSH biosynthesis is unlikely to continue in strain NZ9000(pNZ3203) during acid challenge. The decline of GSHin in NZ9000(pNZ3203) cells was not as large as that in SK11(GSH⫹) cells, suggesting the protective roles of GSH in NZ9000(pNZ3203) and SK11(GSH⫹) cells against acid stress are different. GSH protects GAPDH in L. lactis SK11 but not in L. lactis NZ9000. The third approach we used to gain more insights into the protective role of GSH was to determine the activity of some key enzymes which are physiologically and metabolically essential for the cell. One of the key enzymes that we investigated was GAPDH, which plays a key role in glycolysis. The decrease of GAPDH activity induced by the acidic environment has been shown to be related to the death of acidstressed cells of Lactobacillus (7). An experiment was performed to look for whether the role of GSH against acid stress in L. lactis is related to protection of GAPDH activity. The GAPDH activities of SK11(GSH⫹), SK11(GSH⫺), NZ9000 (pNZ3203), and NZ9000(pNZ8148) cells were investigated under different acid stress conditions (Fig. 5A and B). Significantly greater GAPDH activities were observed in SK11 (GSH⫹) cells and NZ9000(pNZ3203) cells upon challenge at pH 2.5 for 20 min. However, with challenge at pH 4.0, significantly greater GAPDH activities were observed only in SK11 (GSH⫹) cells, not in NZ9000(pNZ3203) cells. This again suggests that the mechanisms behind the protective role of GSH in SK11(GSH⫹) and NZ9000(pNZ3203) cells differ. The other two glycolytic enzymes that we investigated were PK and HK. The activities of these two enzymes in both SK11(GSH⫹) and NZ9000(pNZ3203) cells were not significantly greater than those of the control, suggesting they were not protected by GSH during acid challenge. DISCUSSION The study described here demonstrated that GSH imported by L. lactis subsp. cremoris SK11 or produced by an engineered

strain of L. lactis subsp. cremoris, NZ9000, can protect both strains against a long-term mild acid challenge and a shortterm severe acid challenge. This increases our knowledge of the physiological role of GSH in L. lactis besides its role in protecting the cells from oxidative stress (11, 17). The GSH in SK11(GSH⫹) cells and NZ9000(pNZ3203) cells displayed similar profiles of protection when cells were challenged at pH 2.5 or at pH 4.0 (Fig. 2). However, more-detailed comparative studies suggested the mechanisms behind the protective roles of GSH in these two strains may differ. Investigation of the changes in pHi, GSHin, and GAPDH activity in SK11(GSH⫹) cells allowed us to propose putative protective mechanisms of GSH for strain SK11. One of the proposed mechanisms is prevention of the rapid decline of pHi using GSH as a sacrificial substance. We have shown that SK11 (GSH⫹) cells had a significantly higher pHi than the control when challenged at pH 2.5 for 30 min and at pH 4.0 for longer than 4 h (Fig. 3). A previous study showed that an induced increase of pHi in L. lactis subsp. cremoris 712 protects the cells from lethal acidification (20). We therefore believe the maintenance of a significantly higher pHi in SK11(GSH⫹) cells during acid challenge contributed to the significantly increased survival rate. The presence of GSH itself did not increase the initial pHi, since SK11(GSH⫹) and SK11(GSH⫺) cells had comparable pHis prior to acid challenge (Fig. 3A, 0-h data), so it can be assumed that the addition of GSH did not disturb the intracellular pH homeostasis. However, we observed that most GSH disappeared before severe killing took place (Fig. 4A). The depletion of GSH is not due to the exchange between the reduced-form GSH and the oxidized form, GSSG, since the enzymatic recycling method that we used determines the total GSH concentration (GSH plus GSSG) (29). The disappearance of the intracellular GSH in SK11 cells suggests that GSH might be bound to certain cellular components in an irreversible manner, since the GSH bound to a protein via a disulfide bond (G-S-S-protein) can still be reduced and recovered by the glutathione reductase present in the assay (29) and ultimately detected. Alternatively, GSH may be acting as a trap for acid, which consequently protects vital cellular constituents from

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acid damage. A similar effect has been reported in defending the E. coli cells against chlorine compounds, where the intracellular GSH reacts with the chlorine compounds nonenzymatically and sacrificially (5). However, the exact fate of the disappeared GSH in SK11(GSH⫹) cells upon acid challenge remains unclear at this stage. The second protective mechanism which may take place in SK11(GSH⫹) cells is the protection of GAPDH via S thiolation. The fact that certain irreversible damage can be prevented by protein S thiolation, in which protein SH groups form mixed disulfides with low-molecular-mass thiols, such as GSH (16), has been investigated, and GAPDH has been identified as the major target of S thiolation (26). Improvement of GAPDH activity in the presence of GSH in eukaryotic cells has been reported (13, 22). We postulate that the GAPDH in SK11 (GSH⫹) cells was protected by S thiolation with GSH, which resulted in a slow decrease of GAPDH activity upon acid challenge. The S -thiolation of GAPDH may be related to the decrease of GSHin; however, it may not account for the depletion of GSH, since the S thiolation is reversible. The protective role of GSH against acid stress in strain SK11 is unlikely to be caused by cross-protection, since the presence of GSH in SK11 did not affect growth. However, the final biomass of strain NZ9000(pNZ3203) was approximately 14% lower than that of NZ9000(pNZ8148). This is likely to reflect the protein burden on strain NZ9000(pNZ3203), since two heterogeneous proteins were produced, but the effect of GSH production cannot be excluded. Therefore, the protective mechanism of GSH in NZ9000(pNZ3203) cells seems to be complex. We found that the pHi of NZ9000(pNZ3203) was significantly higher than that of the control (Fig. 3A), which might be the major reason leading to the improved survival upon acid challenge. However, the decrease of GSHin in NZ9000(pNZ3203) cells was much smaller than that in SK11 (GSH⫹) cells, suggesting that the GSH in NZ9000(pNZ3203) was not directly involved in any biological process which may heavily deplete GSH. In addition, no significant difference in GAPDH activity between NZ9000(pNZ3203) and NZ9000 (pNZ8148) was observed when cells were challenged at pH 4.0, while the difference between the two cell types was significant with challenge at pH 2.5 for 20 min. The activity of GAPDH in NZ9000 is about 20% of that in strain SK11 (Fig. 5). This suggests that GAPDH may not be a major target for GSH in NZ9000(pNZ3203) cells when they encounter a mild acid challenge. Essentially, the differences between the protective mechanism of GSH between SK11(GSH⫹) cells and NZ9000 (pNZ3203) cells may be due to the different physiological backgrounds. Strain SK11 can import GSH from the environment, while strain NZ9000 cannot. This implies that the GSH imported by strain SK11 is of physiological significance, and the protective mechanism is therefore relatively easily understood. On the contrary, since GSH is not physiologically essential for strain NZ9000, it is relatively difficult to elucidate the protective mechanism in strain NZ9000(pNZ3203) unless “omics” techniques are applied to map the site where protection takes place. The physiological role of GSH in gram-positive bacteria is poorly understood, since most gram-positive bacteria do not possess the biosynthetic capability. To date, among gram-positive bacteria, only Streptococcus agalactiae (15) and Listeria


monocytogenes (12) were shown experimentally to have GSH biosynthetic capability, but the physiological role of GSH in these two species remains unknown. Although the primary physiological function of GSH is involved in oxidative stress resistance, we show that GSH can protect L. lactis against acid stress. The improvement of acid stress resistance of L. lactis upon introduction of GSH provides a new model with which to investigate the mechanism for acid stress resistance in L. lactis. It also provides a model with which to further investigate the role of GSH in acid stress resistance, which is less well known than the role of GSH in oxidative-stress resistance. Moreover, the results presented here are also of industrial importance, especially in the case of strain SK11, which is widely used by the dairy industry in cheese making (27). Since the growth and storage environments for a starter culture are normally acidic, greater stability of the starter culture can be obtained by selecting a starter strain which can accumulate GSH or by introducing GSH biosynthetic capability from a GRAS (generally regarded as safe) organism into a starter strain using a foodgrade vector. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Sciences Foundation of China (30300009), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-G-005), the National Basic Research Program (2007CB707803), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0532). Y.L. is supported by the Hundreds of Talents Program of the Chinese Academy of Sciences, and J.C. is supported by the National Science Fund for Distinguished Young Scholars of China (20625619). REFERENCES 1. Aust, A., S. Yun, and C. Suelter. 1975. Pyruvate kinase from yeast (Saccharomyces cerevisiae). Methods Enzymol. 42:176–182. 2. Barnard, E. A. 1975. Hexokinase from yeast. Methods Enzymol. 42:6–20. 3. Breeuwer, P., J. Drocourt, F. M. Rombouts, and T. Abee. 1996. A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester. Appl. Environ. Microbiol. 62:178–183. 4. Carmel-Harel, O., and G. Storz. 2000. Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54:439–461. 5. Chesney, J. A., J. W. Eaton, and J. R. Mahoney, Jr. 1996. Bacterial glutathione: a sacrificial defense against chlorine compounds. J. Bacteriol. 178: 2131–2135. 6. Christensen, J. E., E. G. Dudley, J. A. Pederson, and J. L. Steele. 1999. Peptidases and amino acid catabolism in lactic acid bacteria. Antonie Leeuwenhoek 76:217–246. 7. Corcoran, B. M., C. Stanton, G. F. Fitzgerald, and R. P. Ross. 2005. Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl. Environ. Microbiol. 71:3060–3067. 8. Duwat, P., B. Cesselin, S. Sourice, and A. Gruss. 2000. Lactococcus lactis, a bacterial model for stress responses and survival. Int. J. Food Microbiol. 55:83–86. 9. Even, S., N. D. Lindley, and M. Cocaign-Bousquet. 2003. Transcriptional, translational and metabolic regulation of glycolysis in Lactococcus lactis subsp. cremoris MG 1363 grown in continuous acidic cultures. Microbiology 149:1935–1944. 10. Ferguson, G. P. 1999. Protective mechanisms against toxic electrophiles in Escherichia coli. Trends Microbiol. 7:242–247. 11. Fu, R. Y., R. S. Bongers, S. van II, J. Chen, D. Molenaar, M. Kleerebezem, J. Hugenholtz, and Y. Li. 2006. Introducing glutathione biosynthetic capability into Lactococcus lactis subsp. cremoris NZ9000 improves the oxidativestress resistance of the host. Metab. Eng. 8:662–671. 12. Gopal, S., I. Borovok, A. Ofer, M. Yanku, G. Cohen, W. Goebel, J. Kreft, and Y. Aharonowitz. 2005. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. J. Bacteriol. 187:3839–3847. 13. Gregus, Z., and B. Nemeti. 2005. The glycolytic enzyme glyceraldehyde-3phosphate dehydrogenase works as an arsenate reductase in human red blood cells and rat liver cytosol. Toxicol. Sci. 85:859–869.

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