Identification Of Bile Salt-Tolerant Genes In Lactobacillus Using Comparative Genomics

The bile salt tolerance of Lactobacillus is one of crucial factors for its survival and beneficial effects in the intestine.Researches have shown significant differences in the tolerance ability of Lactobacillus,and the mechanisms responsible for this differential phenomenon are not clear.In recent years,researchers have explored potential bile salt tolerance mechanisms and differential genes or differential proteins under bile salt treatment in Lactobacillus using transcriptomics or proteomics.However,these researches only focused on single strains and these predicted differential genes or differential proteins have not been further validated.In this research,based on the differences in bile salt tolerance phenotypes of107 Lactobacillus strains,we investigated the metabolic pathways and functional genes related to bile salt tolerance in Lactobacillus from a population perspective by comparative genomics,and performed functional validation by the means of overexpression and knockout.This research explored the genes related to bile salt tolerance in Lactobacillus and using them as molecular targets for rapid screening of Lactobacillus with bile salt tolerance in the future.The main findings are as follows:(1)The bile salt tolerance of 107 Lactobacillus strains(Limosilactobacillus fermentum,Lactiplantibacillus plantarum and Lacticaseibacillus paracasei are classified as Limosilactobacillus,Lactiplantibacillus and Lacticaseibacillus according to the latest species list.The discussion in this paper follows the classification used at the beginning of the research and describes them as three species.)from different sources(feces,vegetables,and dairy products)showed that there were interspecies and interstrains differences.L.plantarum showed excellent bile salt tolerance,while L.paracasei showed bile salt sensitivity.Under 1.2%bile salt treatment,the growth rates of L.fermentum FBJSY314,L.fermentum FSH101 and L.fermentum FBJSY361 were less than 5%,while the growth rates of L.fermentum 156 and L.fermentum FSCP241 remained were above 70%;the growth rate of L.plantarum RS417 was less than 20%,while the growth rates of L.plantarum RS4,L.plantarum S83,and L.plantarum CCFM239 were over 90%.Correlation analysis showed that the presence of the reported bile salt tolerance gene bsh did not seem to show a correlation with the bile salt tolerance of Lactobacillus in this research.Phylogenetic analysis showed that the bile salt tolerance of Lactobacillus could not be determined from the source of isolation alone,but most of the strains isolated from fermented foods showed bile salt tolerance ability.(2)Comparative genomic analysis based on interspecies difference revealed that L.plantarum is rich in genes encoding carbohydrate-active enzymes and antibiotic transporter proteins,and that the core genome of L.plantarum contains specific amino acid metabolic pathways(β-lactam antibiotic biosynthesis,2-oxocarboxylic acid metabolism),whereas the core genome of L.paracasei lacks vitamin B synthetic pathways(riboflavin metabolism and folate metabolism).The results suggested that interspecies differences of bile salt tolerance in Lactobacillus are related to their utilization of carbon sources.Strains possessing genes encoding multiple carbon source utilization can generate sufficient energy to sustain cell growth under bile salt stress.Comparative genomic results based on interstrains differences showed that the predicted functional genes for bile salt tolerance in L.fermentum were enriched in the phosphotransferase system and the two-component system,whereas those in L.plantarum were enriched in the phosphotransferase system,the ABC transporter protein,the two-component system and the carbohydrate metabolic pathway.(3)Functional validation of genes predicted by comparative genomics was conducted by means of overexpression and knockout.Results of overexpression experiments showed that overexpression of proteins encoded by representative genes(sfc A,ldh A,mtl R,sec B,agrA,and gdh)that were upregulated in relative transcript levels after bile salt treatment in the bile salt-sensitive strain L.plantarum RS417 significantly increased the tolerance ability by 46.42%to 68.01%.Results of knockout experiments showed that the mutation of agrA in L.plantarum S83,a bile salt-tolerant strain,significantly reduced the tolerance of the strain,while the mutation of gdh did not affect the tolerance of the strain.Metabolomic analysis showed that carbohydrate utilization-related metabolites such as pyruvate and 2-ketoglutarate were significantly upregulated after overexpression of the protein encoded by agrA,while the mutation of agrA resulted in significant downregulation of amino acid utilization-related metabolites such as L-glutamine and L-glutamate.This phenomenon implied that agrA associated with the two-component system generates sufficient energy to sustain bacterial growth under bile salt treatment by sensing bile salt signals and regulating pathways such as carbohydrate utilization and amino acid utilization.Specific primers were designed using agrA as a molecular target,and 5 strains of bile salt-tolerant Lactobacillus were screened from16 strains.Correlation analysis showed that the growth rates of the strains under bile salt conditions was correlated with agrA.