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Proteomic analyses to reveal the protective role of glutathione in resistance of Lactococcus lactis to osmotic stress.

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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC2869154 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Proteomic analyses to reveal the protective role of glutathione in resistance of Lactococcus lactis to osmotic stress.

Zhang Yanhe Y Zhang Yanping Y Zhu Yan Y Mao Shaoming S Li Yin Y

Applied and environmental microbiology 20100326 10

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 ...[more]

PMID: 20348298

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Project description:UnlabelledBoth prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 μM. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions.ImportanceUnderstanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.

Project description:BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable genome.

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Proteomic analyses to reveal the protective role of glutathione in resistance of Lactococcus lactis to osmotic stress.

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Proteomic analyses to reveal the protective role of glutathione in resistance of Lactococcus lactis to osmotic stress.

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