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A Lactobacillus plantarum strain with a deletion in the gene rpoN, encoding the alternative sigma factor 54 (σ54), displayed a 100-fold-higher sensitivity to peroxide than its parental strain. This feature could be due to σ54-dependent regulation of genes involved in the peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for σ54. Subsequent experiments revealed that the impaired expression of the mannose phosphotransferase system (PTS) operon in the rpoN mutant caused the observed increased peroxide sensitivity.
The lactic acid bacterium Lactobacillus plantarum is encountered in many dairy, meat, and plant fermentations. Furthermore, it is frequently encountered in the human gastrointestinal tract (1), and some strains are marketed as probiotics (6). During exponential growth, L. plantarum converts glucose almost completely to lactate (8). However, under aerobic conditions, a pathway involving lactate dehydrogenase, pyruvate oxidase, and acetate kinase enzymes can convert lactate to acetate and produces one ATP (10, 11, 15). This pathway produces hydrogen peroxide (H2O2) and carbon dioxide (CO2) as side products, and accumulation of peroxide ultimately leads to aerobic growth arrest (3). Together with superoxide (·O2−) and hydroxyl radicals (·OH), hydrogen peroxide belongs to a group of compounds known as reactive oxygen species (ROS). Hydrogen peroxide is relatively inert toward organic compounds (5), but it reacts readily with metal ions like Fe2+ to yield hydroxyl radicals (Fenton's reaction) that damage DNA, proteins, and membranes (9). Analyses of the genome sequence of Lactobacillus plantarum WCFS1 revealed the presence of a sophisticated defense system against hydrogen peroxide, which includes a putative glutathione peroxidase-encoding gene (gpo) (14). An in silico regulatory network prediction for the alternative sigma factor 54 (σ54) suggested σ54-dependent expression of gpo (19), postulating a role for σ54 in the oxidative stress response of L. plantarum.
In this article, we describe the increased peroxide sensitivity of an L. plantarum rpoN mutant (rpoN::cat; lacking a functional σ54) compared to its parental strain. Subsequent experiments, including transcriptome analyses and oxidative stress tolerance measurement in mannose phosphotransferase system (PTS) deletion strains, revealed that the previously reported impaired expression of the mannose PTS operon in the rpoN mutant (15), rather than the postulated σ54-dependent expression of gpo, is responsible for the observed increased peroxide sensitivity.
To investigate the possible role of σ54 in peroxide stress survival, wild-type and rpoN mutant cells were grown in MRS until an optical density at 600 nm (OD600) of 1.0 was reached, after which hydrogen peroxide was added to a final concentration of 40 mM. Samples were taken after 30 min and immediately diluted in MRS, and CFU were enumerated by plating appropriate dilutions. The 40 mM dosage of hydrogen peroxide required for lethality in L. plantarum appears to be relatively high compared with dosages reported to be lethal for other species, like Lactococcus lactis and Streptococcus pyogenes (13, 16). However, the culture density at which the peroxide stress was applied was significantly lower in those studies than in the work presented here, which may affect the actual concentration experienced by individual cells. In addition, the complexity of MRS medium may add to this difference, since certain compounds present in this medium may scavenge the oxygen radicals derived from hydrogen peroxide, thereby reducing the effective concentration of this stress agent (17). Alternatively, this difference could be explained by the relatively large repertoire of L. plantarum functions that are potentially involved in oxidative stress tolerance, including NADH oxidases, glutathione (GSH) reductases, a GSH peroxidase, NADH peroxidases, and thioredoxins (14).
Due to peroxide treatment, the relative viable count of the wild-type culture appeared to be reduced by approximately 3 orders of magnitude after 30 min, whereas that of NZ7306 (rpoN::cat) was reduced at least 100-fold more (Fig. (Fig.1).1). This increased sensitivity of NZ7306 could be caused by σ54-dependent regulation of genes involved in the oxidative stress-specific response of L. plantarum or genes involved in the general stress response in this organism. To evaluate the latter possibility, the relative capacities of the wild type and its rpoN mutant derivative to survive lethal levels of UV and heat stress were determined, and they did not appear to differ significantly (Fig. (Fig.2).2). Therefore, the reduced stress tolerance observed in NZ7306 (rpoN::cat) appears to be specific for peroxide stress, which would support a regulatory role of σ54 in the control of a candidate peroxide stress tolerance factor, such as glutathione peroxidase.
The role of σ54 in survival of acute peroxide stress in L. plantarum could relate to σ54-dependent adaptation to peroxide stress conditions. To compare peroxide stress adaptation capacity between the wild type and NZ7306, L. plantarum cultures were pretreated with a sublethal peroxide concentration (3.5 mM for 30 min) prior to addition of a lethal peroxide dose. Addition of 3.5 mM hydrogen peroxide resulted in a temporal growth stagnation for approximately 2 h, after which growth resumed, indicating that the concentration of hydrogen peroxide was indeed sublethal. In both strains, adaptation induced an approximate 100-fold-improved relative survival (Fig. (Fig.1),1), suggesting that the adaptation capacity is not affected by the rpoN mutation.
To evaluate the possible involvement of σ54 in transcription of the glutathione peroxidase gene (gpo) or other genes required for peroxide stress survival, full-genome transcriptome analyses were performed as described previously (20). To this end, wild-type strain WCFS1 and its rpoN mutant derivative (Table (Table1)1) were grown to mid-exponential phase (OD600 = 1.0) and treated with 3.5 mM hydrogen peroxide for 30 min. Subsequently, cells were harvested and RNA was isolated for transcriptome profiling (Fig. (Fig.3).3). This profiling revealed only a small difference between the peroxide response of the wild type and that of NZ7306 (rpoN::cat), exemplified by the relatively small list of genes displaying an interaction effect, i.e., a significant differential response to peroxide between NZ7306 and the wild type (Table (Table2).2). The GSH peroxidase gene was expressed at a higher level in both strains during peroxide stress (data not shown), suggesting that the regulation of this gene does not depend on σ54. The ratios of the genes displaying an interaction effect are low, and therefore, the transcriptome analyses failed to disclose direct clues that could explain the increased peroxide sensitivity observed in the σ54 mutant.
The transcriptome analyses showed no significant difference in peroxide exposure between the wild type and NZ7306 (rpoN::cat), raising the question of whether the peroxide sensitivity of NZ7306 is caused by the reported impaired expression of the mannose PTS operon in this strain (19). To investigate the putative role of the mannose PTS in peroxide tolerance, the hydrogen peroxide sensitivities of two strains, NZ7307 and NZ7308, lacking expression of a functional mannose PTS were tested. NZ7307 is mutated in the mannose operon regulator gene manR, whereas NZ7308 harbors a mutation in the transferase-encoding gene manIIC (Fig. (Fig.4;4; Table Table11 ). When these mannose PTS mutants were treated with a lethal dose of hydrogen peroxide, the viable cell counts showed a 1- to 2-order-of-magnitude-higher reduction than the wild type (Fig. (Fig.5),5), which parallels the reduction observed for the rpoN mutant, NZ7306 (Fig. (Fig.1).1). Since NZ7306, NZ7307, and NZ7308 share the lack of expression of a functional mannose PTS, these results seem to point at a direct relation between the presence of a functional mannose PTS and peroxide tolerance in L. plantarum.
The mannose PTS is a major glucose-transporting PTS in various lactic acid bacteria (2), and deletion of this transporter leads to reduced growth in L. plantarum (15), which is probably due to reduced glucose uptake capacity. Reduced glucose uptake in cells lacking a functional mannose PTS could lead to a reduction in the energy generation rate, which may cause increased peroxide sensitivity. However, this “lack-of-energy” explanation would also predict an increased sensitivity to other forms of stress (e.g., UV, heat, etc.), which could not be experimentally confirmed. Comparative analysis of mannose PTSs suggests a relatively late evolutionary origin of this transport system (21) and phylogenetic profiling placed the Escherichia coli and L. plantarum mannose PTSs in the same highly conserved group of mannose transporters (21). The mannose PTS homolog in E. coli is highly resistant to oxidizing agents (12), and the close relationship between the systems suggests a similar robustness of the L. plantarum system. This suggests the maintenance of the glucose import function of the mannose PTS during peroxide exposure, while the alternative transporters that are used for glucose import in the mutant strains (NZ7306, NZ7307, and NZ7308) are inactivated under these conditions. Consequently, the cells that lack a mannose PTS will have major problems in energy generation processes that are required to launch an appropriate peroxide-induced stress response, ultimately leading to increased peroxide sensitivity. Overall, our results indicate a role of the mannose PTS in oxidative stress tolerance in L. plantarum and corroborate the previously observed resistance to oxidizing agents of this family of transport systems.
Oxidative stress is an industrially relevant stress condition, which may be encountered during processing or as a consequence of hydrogen peroxide production as a side product of carbohydrate fermentation. Therefore, our findings imply that specific culture conditions that induce expression of the mannose PTS (i.e., growth on specific carbon sources like glucose or mannose ) allow the production of bacterial cells displaying increased oxidative stress tolerance, which is relevant for starter-culture production and fermentation industries.
The microarray design was submitted to the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under GEO accession number GPL6368. Primary transcriptome data were submitted to GEO under accession number GSE-11351.
This work was supported by grant no. IGE1018 from the Dutch IOP-Genomics Program.
Published ahead of print on 2 April 2010.