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Lactobacillus plantarum, commonly isolated from plant material, is widely used to produce various types of fermented foods. However, nutrient-limiting conditions are often encountered during industrial applications. The present study aimed to investigate the response of L. plantarum P-8 to glucose-limited conditions in a long-term experiment. Genotypic and proteomic changes in L. plantarum P-8 were monitored over 3 years in glucose-limited and glucose-normal media using whole-genome resequencing and tandem mass tag-based quantitative proteomic analysis. Results showed that L. plantarum employed numerous survival mechanisms, including alteration of the cell envelope, activation of the PTS system, accumulation and consumption of amino acids, increase in the metabolism of carbohydrates (via glycolysis, citric acid cycle, and pyruvate metabolism), and increase in the production of ATP in response to glucose starvation. This study demonstrates the feasibility of experimental evolution of L. plantarum P-8, while whole-genome resequencing of adapted isolates provided clues toward bacterial functions involved and a deeper mechanistic understanding of the adaptive response of L. plantarum to glucose-limited conditions., Significance: We have conducted a 3-year experiment monitoring genotypic and proteomic changes in Lactobacillus plantarum P-8 in glucose-limited and glucose-normal media. Whole-genome resequencing and tandem mass tag-based quantitative proteomics were performed for analyzing genomic evolution of L. plantarum P-8 in glucose-limited and glucose-normal conditions. In addition, differential expressed proteins in all generations between these two conditions were identified and functions of these proteins specific to L group were predicted. L. plantarum employed numerous survival mechanisms, including alteration of the cell envelope, activation of the PTS system, accumulation and consumption of amino acids, increase in the metabolism of carbohydrates (glycolysis, citric acid cycle, and pyruvate metabolism), and increase in the production of ATP in response to glucose starvation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nutrient starvation is an important survival challenge for bacteria during industrial production of functional foods. As next-generation sequencing technology has greatly advanced, we performed proteomic and genomic analysis to investigate the response of Lactobacillus casei Zhang to a glucose-restricted environment. L. casei Zhang strains were permitted to evolve in glucose-restricted or normal medium from a common ancestor over a 3 year period, and they were sampled at 1000, 2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations and subjected to proteomic and genomic analyses. Genomic resequencing data revealed different point mutations and other mutational events in each selected generation of L. casei Zhang under glucose restriction stress. The differentially expressed proteins induced by glucose restriction were mostly related to fructose and mannose metabolism, carbohydrate metabolic processes, lyase activity, and amino-acid-transporting ATPase activity. Integrative proteomic and genomic analysis revealed that the mutations protected L. casei Zhang against glucose starvation by regulating other cellular carbohydrate, fatty acid, and amino acid catabolism; phosphoenolpyruvate system pathway activation; glycogen synthesis; ATP consumption; pyruvate metabolism; and general stress-response protein expression. The results help reveal the mechanisms of adapting to glucose starvation and provide new strategies for enhancing the industrial utility of L. casei Zhang.

Background: The extensive use of antibiotics in medicine has raised serious concerns about biosafety. However, the effect of antibiotic application on the adaptive evolution of microorganisms, especially to probiotic bacteria, has not been well characterized. Thus, the objective of the current work was to investigate how antibiotic selection forces might drive genome adaptation using Lactobacillus (L.) casei Zhang as a model., Methods: Two antibiotics, amoxicillin and gentamicin, were consistently applied to the laboratory culture of L. casei Zhang. We then monitored the mutations in the bacterial genome and changes in the minimum inhibitory concentrations (MICs) of these two antibiotics along a 2000-generation-cultivation lasted over 10 months., Results: We found an approximately 4-fold increase in the genome mutation frequency of L. casei Zhang, i.e. 3.5 × 10 -9 per base pair per generation under either amoxicillin or gentamicin stress, when compared with the parallel controls grown without application of any antibiotics. The increase in mutation frequency is significantly lower than that previously reported in Escherichia (E.) coli. The rate of de novo mutations, i.e. 20 per genome, remained low and stable throughout the long-term cultivation. Moreover, the accumulation of new mutations stopped shortly after the maximum bacterial fitness (i.e. the antibiotic MICs) was reached., Conclusions: Our study has shown that the probiotic species, L. casei Zhang, has high genome stability even in the presence of long-term antibiotic stresses. However, whether this is a species-specific or universal characteristic for all probiotic bacteria remains to be explored.