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Appl Environ Microbiol. 2010 May; 76(10): 3177–3186.
Published online 2010 March 26. doi:  10.1128/AEM.02942-09
PMCID: PMC2869154

Proteomic Analyses To Reveal the Protective Role of Glutathione in Resistance of Lactococcus lactis to Osmotic Stress[down-pointing small open triangle]

Yanhe Zhang,1,3, Yanping Zhang,1, Yan Zhu,1,2 Shaoming Mao,1,2 and Yin Li1,*

Abstract

Previously, we have shown that glutathione can protect Lactococcus lactis against oxidative stress and acid stress. In this study, we show that glutathione taken up by L. lactis SK11 can protect this organism against osmotic stress. When exposed to 5 M NaCl, L. lactis SK11 cells containing glutathione exhibited significantly improved survival compared to the control cells. Transmission electron microscopy showed that the integrity of L. lactis SK11 cells containing glutathione was maintained for at least 24 h, whereas autolysis of the control cells occurred within 2 h after exposure to this osmotic stress. Comparative proteomic analyses using SK11 cells containing or not containing glutathione that were exposed or not exposed to osmotic stress were performed. The results revealed that 21 of 29 differentially expressed proteins are involved in metabolic pathways, mainly sugar metabolism. Several glycolytic enzymes of L. lactis were significantly upregulated in the presence of glutathione, which might be the key for improving the general stress resistance of a strain. Together with the results of previous studies, the results of this study demonstrated that glutathione plays important roles in protecting L. lactis against multiple environmental stresses; thus, glutathione can be considered a general protectant for improving the robustness and stability of dairy starter cultures.

Lactic acid bacteria (LAB) are widely used in the food and fermentation industries (5). During the manufacturing process, LAB often encounter various environmental stresses, including acid stress, oxidative stress, heat stress, and cold stress. The physiological responses of LAB to these environmental stresses have been extensively studied and characterized (10, 16, 20, 24, 25, 35, 42, 45, 47). In addition, LAB are often challenged with high salt concentrations during cheese fermentation (11), vegetable pickling (39), food preservation (17), and fermentative production of lactic acid (4). The osmotic stress triggered by high salt concentrations results in both structural and physiological injury of cells (8, 40). Therefore, the ability of LAB to survive, grow, and metabolize actively under osmotic stress conditions is very important in industry (53).

Bacteria have evolved various mechanisms to survive osmotic challenge. When there is a hyperosmotic shock, some bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, and Lactococcus lactis) exhibit plasmolysis due to a rapid efflux of water to balance the change in osmotic pressure (8, 9). The accumulated ions are then exchanged by osmoprotectants, such as amino acids (e.g., glutamate [8, 44] and proline [8, 9, 36]) and quaternary amines (e.g., glycine betaine [36, 37, 48]). Various proteins contribute to the accumulation of osmoprotective substances during osmotic stress (8, 9, 36, 37). Proteomics has been used to investigate the effect of osmotic challenge on Bacillus subtilis (1, 21), Streptococcus mutans (46), Synechocystis sp. strain PCC 6803 (15), Aspergillus nidulans (23), L. lactis (22), and Halobacterium salinarum (27). Several osmotic stress-specific proteins, including enzymes involved in proline biosynthesis in B. subtilis, glucosylglycerol biosynthesis in Synechocystis sp., and glycerol biosynthesis in A. nidulans, have been identified. In these species, a set of proteins responding to both heat shock and salt stress were found. Moreover, gene expression responses to osmotic stress in L. lactis were studied at the transcriptional level, and proteins responding to heat, acid, and osmotic stress were found (51).

Glutathione (γ-glytamylcysteinylglycine [GSH]) is the most important nonprotein thiol compound in living organisms (34). Although the primary physiological role of GSH is a role in resistance to oxidative stress, this molecule also has a role in dealing with osmotic stress in E. coli (44). Our previous studies have shown that GSH can protect an L. lactis strain against oxidative stress and acid stress (14, 29, 52), suggesting that it might have multiple functions in LAB. We therefore wondered if GSH is able to protect LAB against osmotic stress. Moreover, we wanted to obtain more insight into the protective role of GSH in LAB, which is currently largely unknown. To this end, we used L. lactis subsp. cremoris SK11 as a model LAB strain and investigated whether the GSH taken up by strain SK11 can protect this organism against osmotic stress. Since the genome of strain SK11 is now available (31) and the industrial relevance of this strain is understood at the genome level (54), comparative proteomic analyses using strain SK11 cells containing or not containing GSH that were exposed or not exposed to osmotic stress were performed with the aim of determining the protective role of GSH in the resistance of L. lactis to osmotic stress.

MATERIALS AND METHODS

Strains and culture conditions.

L. lactis subsp. cremoris SK11 was the strain used in our previous studies (29, 52). A culture was transferred from a −70°C frozen stock to M17 broth (Merck, Germany) containing 5 g/liter lactose (LM17) and incubated at 30°C for 16 h to obtain a preculture. The preculture was then inoculated (1%, vol/vol) into chemically defined medium (CDM) (29) supplemented or not supplemented with 3.2 mM GSH and incubated statically at 30°C for 16 h. The SK11 cells containing and not containing intracellular GSH were designated GSH+ cells and GSH cells, respectively.

Osmotic challenge.

L. lactis SK11 GSH+ and GSH cells were harvested from 10-ml cultures (optical density at 600 nm [OD600], approximately 2.0) by centrifugation at 10,000 × g and 4°C for 10 min. The cell pellets were washed twice with saline (0.85% NaCl) to remove the residual medium and then resuspended in 10 ml 5 M NaCl to start an osmotic challenge. To determine the time course of cell survival, 1-ml samples were removed, and the cells were pelleted by centrifugation at 10,000 × g for 5 min, washed with saline to remove the residual NaCl, and then resuspended in 1 ml saline. Ten-microliter serially diluted samples were spotted onto LM17 agar plates in triplicate and incubated at 30°C for 24 h. The rates of survival of cells (expressed as percentages) were calculated by dividing the number of CFU of stressed cells by the number of CFU of cells before stress exposure.

Morphology.

Transmission electron microscopy (TEM) was used to analyze the morphology of stressed lactococcal cells. Cells were first fixed by adding 2.5% (vol/vol) glutaraldehyde and incubating the culture overnight; then the cells were pelleted by centrifugation at 10,000 × g and 4°C for 5 min, and the supernatant was removed. The fixed sample was then dehydrated with ethanol (30%, 50%, 70%, 85%, and 95%) and embedded in resin, which allowed thin sectioning and provided resistance to the TEM environment. Furthermore, each section was impregnated with heavy metals using procedures described previously (32) in order to increase electron scattering and to enhance the contrast. Prepared cell sections were examined with a transmission electron microscope (JEM-1400; JEOL Ltd., Japan) operating at an acceleration voltage of 80 kV. High-contrast resolution was ensured for 0.38-nm point-to-point and 0.2-nm lattice images.

Preparation of cytoplasmic protein extracts and comparative proteomic analysis.

Preparation of cytoplasmic protein extracts and two-dimensional gel electrophoresis (2-DE) were performed as described by Roy et al. (41). Briefly, samples subjected to various treatments were harvested by centrifugation (5,000 × g for 10 min), washed with 0.85% NaCl twice, resuspended, and sonicated. Each resulting lysate was centrifuged, and then the cell-free supernatant was ultracentrifuged (17,000 × g for 40 min) to remove the membrane fraction, and the reserved supernatant of the cytoplasmic protein fraction was used for proteomic analysis. After dilution in 3 volumes of ice-cold acetone and incubation at −20°C for 16 h, proteins were sedimented by centrifugation at 10,000 × g and 4°C for 30 min. The protein pellets were solubilized in sample lysis buffer containing 8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% Pharmalyte (pH 4 to 7), and 1% (wt/vol) dithiothreitol. Protein concentrations were measured using the Bradford procedure with bovine serum albumin as the standard.

Approximately 500 μg extracted proteins from various samples of strain SK11 was resuspended in a rehydration solution (8 M urea, 2 M thiourea, 4% CHAPS, 0.2% Pharmalyte [pH 4 to 7], 0.001% bromophenol blue) and subjected to 2-DE using IPG strips (GE Healthcare, Uppsala, Sweden). Isoelectric focusing (IEF) of IPG strips was performed with an Ettan IP Gphor 3 system (GE Healthcare, Uppsala, Sweden) using the following stepwise voltage gradient: rehydration at 30 V for 12 h, 200 V for 4 h, voltage increased to 1,000 V within 4 h, voltage increased to 10,000 V within 4 h, and finally voltage kept at 10,000 V for 4 h until the value reached 65,000 V·h. The temperature was maintained at 20°C. After equilibrium was reached, the IPG strips were placed on top of 12.5% polyacrylamide vertical gels, and SDS-PAGE was performed at 16°C and 1 W/gel for 1 h, followed by 10 W/gel, until the bromophenol blue reached the bottom of the gels. Protein spots on the gels were stained with 0.1% Coomassie blue R-250 (Amresco, Solon, OH). Each set of samples was examined independently in triplicate.

The experimental gels were scanned at a resolution of 300 dots per inch (dpi) by using Image Scanner III (GE Healthcare, Uppsala, Sweden), and a comparative analysis of protein spots was performed by using the 2-D Image Master software (GE Healthcare, Uppsala, Sweden). Only protein spots showing consistent and reproducible changes in protein abundance were considered biomarkers associated with multiple experimental tests. A statistical analysis was performed using Student's t test, and P values of <0.05 were considered statistically significant. The relative levels of expression of significantly changed protein spots were calculated by comparing the normalized volumes of the protein spots.

Coomassie blue-stained protein spots with significant changes were excised using gel plugs, transferred to Eppendorf tubes, and then digested and rehydrated in 500 μl of 50 mM NH4HCO3 (pH 8.0) and 20 μl of 10 ng/μl proteomics sequencing-grade trypsin at 37°C for 16 h. Supernatants (0.5 μl) were spotted directly onto a matrix-assisted laser desorption ionization (MALDI) plate for identification of proteins by mass spectrometry as described previously (49). The masses of tryptic peptides were determined with an Applied Biosystems 4700 MALDI-time of flight (TOF)/TOF proteomics analyzer (Applied Biosystems, Framingham, MA). Database data were analyzed by using the MASCOT 2.0 search engine (Matrix Science, London, United Kingdom) to search the L. lactis protein sequence data (20,154 sequences; 576,0076 residues) in the NCBI database, a reference proteomic map (18), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/).

Analysis of gene expression by quantitative reverse transcription-PCR (RT-PCR).

L. lactis SK11 GSH+ and GSH cells exposed or not exposed to osmotic stress were harvested by centrifugation at 5,000 × g at 4°C for 10 min. Cell pellets were pulverized in a mortar under liquid nitrogen conditions to inhibit the intracellular RNase activity. Total RNA from 1 × 106 stressed or unstressed SK11 cells was extracted by using an RNA extraction kit (BioTeke Corporation, China) according to the manufacturer's recommendations. RNA purity was determined by determining the ratio of absorbance at 260 to absorbance at 280 (range, 1.8 to 2.0) with a Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies LLC, Wilmington, DE), and RNA integrity was assessed by electrophoresis. Contaminating genomic DNA in the total RNA was carefully removed by treatment with RNase-free DNase (Promega, Madison, WI) at 37°C for 60 min, and the resulting RNA was used to synthesize cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, CA). Controls that did not contain the reverse transcriptase were included for all samples to verify that genomic DNA was not present.

To evaluate the expression of the genes encoding significantly upregulated and downregulated proteins at the transcription level, real-time RT-PCR was carried out using Power SYBR green PCR master mixture (Applied Biosystems, CA). Reactions (final reaction mixture volume, 25 μl) were performed with an ABI 7000 real-time RT-PCR apparatus (Applied Biosystems, CA), using the following default thermocycler program for all genes: 10 min of preincubation at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. In the last step, a temperature gradient from 95°C to 60°C was used for analysis of dissociation curves. The melting curves for each PCR were analyzed to verify the absence of nonspecific amplification in PCR products. All samples were run in triplicate, and the mean was used for further calculations. All gene expression was normalized using the expression of the internal control gene (16S rRNA gene). The primers used for real-time RT-PCR assays are listed in Table Table11.

TABLE 1.
Primers used in the real-time PCR assay

RESULTS

GSH protects L. lactis against osmotic stress.

Our previous study showed that L. lactis subsp. cremoris SK11 is capable to taking up GSH but is unable to synthesize it (29). We therefore cultivated strain SK11 in CDM supplemented or not supplemented with GSH to obtain SK11 cells containing or not containing GSH. Addition of GSH to CDM does not affect cell growth, as shown in our previous study (29). SK11 GSH+ and GSH cells harvested at stationary phase (16 h) were exposed to an osmotic challenge (5 M NaCl). Two-log and 3-log differences in survival between the GSH+ and GSH cells were observed when cells were challenged with 5 M NaCl for 24 h and 48 h, respectively (Fig. (Fig.1),1), indicating that the presence of intracellular GSH allows L. lactis SK11 cells to resist osmotic stress. Notably, the rate of death of the GSH+ cells during the first 24 h of exposure was much lower than that of the GSH cells (Fig. (Fig.1);1); 9.6% of the GSH+ cells survived the first 24 h of exposure, compared to 0.2% of the GSH cells. This suggests that cellular stability can be maintained better in the presence of GSH when a strong osmotic shock is encountered.

FIG. 1.
Effect of exposure to 5 M NaCl on the survival of stressed L. lactis SK11 GSH+ cells (filled circles) and GSH cells (filled squares). The initial cell concentrations were 2.3 × 109 ± 0.4 × 109 CFU/ml. The natural ...

Morphological changes associated with osmotic stress.

Osmotic stress often leads to cell shrinkage and plasmolysis (8, 9). We therefore compared the morphological changes in SK11 GSH+ and GSH cells before and after treatment with 5 M NaCl by using transmission electron microscopy (Fig. (Fig.2).2). As no significant morphological difference between the unstressed GSH+ and GSH cells prior to exposure to osmotic stress was observed, only the morphology of the unstressed GSH cells is shown in Fig. Fig.2.2. When GSH cells were resuspended in saline for 2 h and 24 h, there was not a significant difference in cell integrity (Fig. (Fig.2A).2A). When GSH cells were exposed to 5 M NaCl for 2 h, the effect on cell integrity was significant (Fig. (Fig.2B).2B). Intracellular components became aggregated and disordered compared with the relatively regular distribution of ribosomes in chromatin in unstressed cells shown previously (28). Electrolucent cavities, which have been called “nuclear vacuoles” previously (12), were observed in a large proportion of GSH cells. Prolonging the exposure of GSH cells to osmotic stress resulted in autolysis of cells (Fig. (Fig.2B,2B, 24 h/25,000× panel). In contrast, the integrity of GSH+ cells was maintained when the cells were exposed to 5 M NaCl for 2 h, and the disordered “nuclear vacuole-like” intracellular structures started to appear only when the cells were exposed for 24 h or longer (Fig. (Fig.2C).2C). This suggests that the L. lactis SK11 cells were well protected by GSH during the first 24 h of exposure, which is in accordance with the high proportion of GSH+ cells that survived the osmotic stress.

FIG. 2.
Effect of GSH on morphological changes in SK11 cells after exposure to osmotic stress. (A) Control, unstressed GSH cells. In the 25,000× images, the dark particles are ribosomes and the ribosome-free spaces contain chromatin (28). (B) ...

Comparative proteomics analyses revealing the protective role of GSH.

Comparative proteomic analyses were performed to determine how GSH protects cells from osmotic stress. We compared the proteomic profiles of L. lactis SK11 GSH+ and GSH cells exposed and not exposed to osmotic stress (designated GSH, GSH+, stressed GSH, and stressed GSH+ cells). Approximately 320 protein spots were identified in the 2-DE gels (Fig. (Fig.3),3), and 33 spots with a difference in abundance of ≥1.5-fold were identified with a MALDI-TOF mass spectrometer. The functions of the corresponding 29 proteins were assigned based on the L. lactis reference proteomic map (18) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/). As shown in Table Table2,2, most of the differentially expressed proteins fell into the following four categories: (i) glycolysis and pyruvate metabolism; (ii) carbohydrate metabolism; (iii) peptidoglycan and purine biosynthesis; and (iv) translation, posttranslational modification, and stress-related proteins. Most interestingly, we found that 21 of the 29 differentially expressed proteins were involved in various metabolic pathways. We therefore summarized the findings for these metabolism-related proteins (Fig. (Fig.4),4), which allowed us to better understand the potential physiological significance of the changes at the protein level.

FIG. 3.
Locations of differentially expressed proteins in gels for cells in the presence or absence of GSH that were exposed or not exposed to osmotic stress. Differentially expressed proteins are labeled, and details about them are shown in Table Table ...
FIG. 4.
Schematic overview of the locations of the differentially expressed proteins in the metabolic pathways of L. lactis SK11 cells in the presence or absence of GSH that were exposed or not exposed to osmotic stress. Upregulated proteins are indicated by ...
TABLE 2.
Differentially regulated proteins responding to osmotic stress and/or GSH

Changes in proteins responding to the presence of GSH.

Previously, we showed that addition of GSH does not affect cell growth (29). Comparative proteomic analysis of unstressed GSH and GSH+ cells (Fig. (Fig.3A3A and and3B)3B) showed that the levels of 24 proteins in the L. lactis SK11 GSH+ cells were significantly altered compared with the levels in the GSH cells (Table (Table2),2), suggesting that the presence of intracellular GSH affected cellular physiology. Sixteen of the 24 proteins were upregulated, whereas 8 proteins were downregulated. Notably, many enzymes involved in glycolysis, including Pfk, FbaA, TpiA, and Pyk, as well as another major protein involved in lactococcal fermentation, lactate dehydrogenase (LDH), were upregulated in GSH+ cells. In addition, glyceraldehyde-6-phosphate 1-dehydrogenase (G6PDH) involved in the pentose phosphate pathway, FemD and MurC involved in peptidoglycan biosynthesis, three proteins involved in stress resistance (DnaK, ClpE, and a DNA-binding ferritin-like protein), one translation protein (PheT), and two posttranslational modification proteins (trypsin-like serine protease and oligopeptidase O1), were found to be upregulated in GSH+ cells.

Several proteins involved in carbohydrate metabolism (LacA, LacB, and LacD) and AldC (alpha-acetolactate decarboxylase) were downregulated in GSH+ cells. In addition, purine biosynthesis-related proteins PurH, GlyA, Fmt, and Gmk were all downregulated, although PurC was highly upregulated (Fig. (Fig.4A4A and and4C4C).

In agreement with previous studies (22, 49), two genes (gapA and gapB) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are present in the genome of L. lactis. However, only GapB was detected on the pH 4 to 7 gel and was present as two major isoforms (Fig. (Fig.3,3, spots 1a and 1b). Although the total abundance of GapB was not obviously altered in the presence of GSH, changes in the two isoforms were observed, suggesting that GSH induced a physiologically relevant posttranslational modification of the enzyme.

Changes in proteins responding to osmotic stress.

Changes in the proteomic profile of LAB responding to low concentrations (33) or to short-term exposure (22) to osmotic stress have been reported previously. To consider the osmotic stress in real microbial processes, we studied the proteins responding to long-term exposure to hyperosmotic stress by performing a comparative proteomic analysis of unstressed GSH cells (Fig. (Fig.3A)3A) and stressed GSH cells (Fig. (Fig.3C).3C). Fifteen differentially expressed proteins were identified after exposure to 5 M NaCl (Table (Table2).2). Of these 15 proteins, 8 were upregulated, whereas 7 were downregulated. Although Pmg (phosphoglycerate mutase) was upregulated in stressed GSH cells, the key glycolytic protein GAPDH (18) and FbaA were downregulated. Three other carbohydrate metabolism-related proteins (LDH, Pmi, and G6PDH) were all downregulated. This suggests that the carbohydrate metabolism of L. lactis is impaired when this organism is exposed to osmotic stress. Surprisingly, two proteins involved in purine and one-carbon folate metabolism (PurH and Fmt) and two proteins involved in peptidoglycan biosynthesis (FemD and GlmS) were upregulated, suggesting that there is a carbon shift from glycolysis to sugar nucleotide biosynthesis and purine biosynthesis following osmotic challenge. Osmotic stress also triggered upregulation of stress proteins, including DnaK and ClpE and two translation proteins (Fmt and PheT).

Changes in proteins responding to the coupled effects of GSH and osmotic stress.

Comparative proteomic analysis of stressed GSH+ cells (Fig. (Fig.3D)3D) and unstressed GSH cells (Fig. (Fig.3A)3A) might reveal the role of GSH in resistance to osmotic stress. As shown in Table Table2,2, 11 proteins were upregulated and 10 proteins were downregulated compared to the control GSH cells. Glycolytic proteins Pfk, FbaA, Tpi, and GapB and proteins involved in peptidoglycan biosynthesis (FemD and GlmS) were upregulated in stressed GSH+ cells. This pattern is similar, but not identical, to the pattern of the GSH+ cells. Proteins involved in purine and one-carbon folate metabolism of stressed GSH+ cells exhibited similar downregulation trends compared to the proteins in unstressed GSH+ cells. Proteins involved in galactose metabolism and pentose phosphate pathways (LacD, AldC, Pmi, and G6PDH) were downregulated in stressed GSH+ cells. Stress protein DnaK was downregulated, whereas the abundance of another stress protein, ClpE, was not altered significantly in the presence of both GSH and osmotic stress.

Verification at the transcription level.

To investigate whether the results for the proteins showing altered levels on 2-DE gels are in good accordance with the changes at the transcription level, six genes (pfk, fbaA, ldh, glmS, purC, and fmt) were randomly selected to test alteration of expression at the transcription level. As shown in Fig. Fig.5,5, real-time RT-PCR experiments showed that transcription of all of these genes (except the fmt gene) was altered in a manner similar to the way in which the level of protein expression was altered, which confirmed the results of the comparative proteomic analysis described above.

FIG. 5.
Comparison of the expression of six differentially expressed proteins at the protein level (three columns on the left in each panel) as revealed by proteomic analysis and at the transcription level (three columns on the right in each panel) as revealed ...

DISCUSSION

As one of the important industrial LAB, L. lactis subsp. cremoris SK11 is widely used in cheese fermentation and dairy processes (2, 7, 43). Although L. lactis SK11 is not able to synthesize and metabolize GSH, the ability of this strain to take up GSH from the environment makes it an ideal model to study the role of GSH. Previously, we have shown that GSH taken up by L. lactis SK11 cells can protect the cells against oxidative stress (29) and acid stress (52). As GSH is present in rich medium and milk, the protective role of GSH in L. lactis strains capable of taking up GSH is therefore relevant for industry. However, information concerning the molecular basis of the role of GSH in protection of L. lactis is still not available.

In this study, we discovered that GSH protected L. lactis against osmotic stress. Moreover, we found that GSH could protect L. lactis SK11 against UV stress and solvent stress (unpublished data). The ability of L. lactis cells to cope with multiple environmental stresses in the presence of GSH suggests that GSH may increase the overall fitness of these cells. Using a comparative proteomic analysis approach, we identified the proteins regulated by GSH and/or by exposure to osmotic stress. The most striking finding is that 17 of 24 proteins differentially expressed in the presence of GSH are involved in metabolic pathways, especially sugar metabolism. Significant upregulation of several key glycolytic enzymes was observed in GSH+ cells (Fig. (Fig.4C).4C). This has great physiological significance, as osmotic stress triggered downregulation of glycolytic enzymes (Fig. (Fig.4B).4B). Microbial adaptation to a high-salinity environment is an energy-consuming process (38). As a fermentative bacterium, L. lactis is expected to derive energy principally from substrate phosphorylation (18, 50). The GSH-induced upregulation of glycolytic enzymes might enable the cells to cope with harsh conditions if a carbon source is available.

Proteins involved in purine biosynthesis, such as PurH, Gmk, and GlyA, were downregulated in GSH+ cells, whereas PurC was upregulated. The upregulation of PurC might enable the cells to produce purines when they are needed. Glycolytic enzymes, such as GAPDH and Pmg, are able to respond to various stresses (3, 22, 26, 40). GAPDH is the major control point for glycolytic flux in LAB (18). In a previous study (52), we observed that the GAPDH activity of L. lactis SK11 GSH+ cells declined slower than that of GSH cells upon exposure to acid stress. This can now be interpreted as GSH-induced upregulation of GAPDH that compensated for the stress-induced decrease in GAPDH activity (Fig. (Fig.4D).4D). Moreover, we found that Pmg was upregulated under osmotic stress conditions, in contrast to the downregulation under low-pH conditions (3, 26). This suggests that LAB might use different energy metabolism strategies to cope with osmotic stress and acid stress.

Upregulation of glycolytic proteins might also result in an increased NADH/NAD+ ratio. Related to this, the upregulation of NADH-dependent LDH could be physiologically significant. The upregulation of LDH (LACR_1455) coincided with the upregulation of Pyk (LACR_1456) and Pfk (LACR_1457). These three proteins are encoded by genes located on the same operon (las operon). As the las operon is activated by the regulatory protein CcpA (30), the overall upregulation of LDH, Pyk, and Pfk in GSH+ cells suggests that CcpA might play an activating role. LDH was downregulated in stressed GSH cells (Fig. (Fig.4B)4B) and upregulated in unstressed GSH+ cells (Fig. (Fig.4C),4C), and its expression was not altered in stressed GSH+ cells (Fig. (Fig.4D);4D); this suggests that the GSH-induced upregulation of LDH could compensate for the stress-induced downregulation of LDH, similar to the findings for GAPDH.

Proteins involved in metabolism of other sugars, including galactose and mannose, were downregulated in GSH+ cells and stressed GSH+ cells. The downregulation of galactose-6-phosphate isomerase subunit LacA (LACR_D06) and subunit LacB (LACR_D05) and tagatose-1,6-diphosphate aldolase LacD (LACR_D03) in GSH+ cells (Table (Table2)2) suggests that the ability of GSH+ cells to assimilate lactose was impaired upon exposure to osmotic stress. These three proteins are encoded on the 47.2-kb plasmid of strain SK11 (GenBank accession number CP000429), which is the second largest of the five plasmids present in strain SK11 (54) and encodes the lactose transport and utilization machinery. In line with the upregulation of glycolytic enzymes, the downregulation of these three proteins in GSH+ cells suggests that GSH might be able to regulate the preference for using carbon sugars for possible adaptation to harsh conditions. GSH allows L. lactis to select and protect the most efficient sugar metabolic pathway, glycolysis, to ensure that energy can be obtained readily when harsh conditions are encountered.

DnaK and ClpE, which were upregulated in unstressed GSH+ cells (Table (Table2),2), have been reported to be induced by salt stress, mild acid treatment, and UV irradiation in LAB (6, 10, 19). Moreover, the DNA-binding ferritin-like protein, a member of the bacterial Dps family that can form DNA-Dps cocrystals to protect DNA from damaging environments (13), was upregulated in unstressed GSH+ cells. This protein might contribute to the morphological difference between stressed GSH+ and stressed GSH cells (13, 28). Heat shock proteins GroEL and GroES were previously found to be induced by heat stress and/or osmotic stress (22). However, the abundance of GroELS proteins did not change in this study. This could be because the samples that we used for proteomic analyses were stressed for 2 h, while the cells used previously were stressed for only 15 min (22).

In conclusion, the induction of energy- and stress-related proteins in the presence of GSH gave L. lactis cells the robustness and fitness to survive multiple environmental stresses. Consistent with the improved availability of energy and induction of stress-related proteins, GSH protects L. lactis against osmotic stress through cooperative induction of adaptive proteins and regulation of energy metabolism-related proteins. Although only a fraction of proteins induced in response to osmotic stress and GSH were identified by comparative proteomic analysis, our findings provide novel insights into the physiological role of GSH in Gram-positive bacteria. Future studies examining how GSH is transported and how glycolytic proteins are regulated in the presence of GSH should improve our understanding, thus helping us to develop strategies to improve the robustness and stability of dairy starter cultures.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (grant 30870040), the National Basic Research Program of China (grant 973, 2007CB707803), and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant KSCX2-YW-G-005). Yin Li was supported by the Hundreds of Talents Program of the Chinese Academy of Sciences.

We thank Jie Zhou and Linjiang Zhu for helpful discussions and Yang Zhu for critically reading the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 26 March 2010.

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