Study On The Molecular Mechanism Of Adaptation Of Lactobacillus Casei Zhang To Gentamycin

Lactobacillus casei Zhang is a probiotic strain that was isolated from koumiss,a traditional fermented milk product,and it has high-level resistance to acid and bile stresses.With the extensive use of antibiotics,it has become a new environmental stress,which is often faced by probiotics.In this study,the gentamycin-driven adaptive strain of L.casei Zhang,Zhang-G-1200,isolated from a prolonged cultivation experiment,was used.This strain was selected for the study because of its gentamycin resistance.Firstly,we analyzed the growth performance of this strain and its parental strain;Secondly,we compared the global protein expression profiles of two strains by an iTRAQ-based proteomic approach.We then verified the biological functions of key proteins through a gene knockout technology.The results are as follows:（1）The analysis results showed that the growth performance of Zhang-G-1200 was completely different from that of the parental strain.The viable counts and OD₆₀₀00 values of Zhang-G-1200 increased more rapidly than that of the parental strain and Zhang-G-1200 had a higher fermenting rate.（2）Proteomic analysis showed that of the 108 differentially expressed proteins screened from the Zhang-G-1200,72 were significantly up-regulated（>2.0-fold,P<0.05）,while 32 proteins were significantly down-regulated（<-2.0-fold,P<0.05）.The function and metabolic pathways of differentially expression proteins were annotated through COG and KEGG.Many differentially expression proteins were stress-related proteins,and other involved in some carbohydrates,amino acids,and purine metabolic pathways,and so on.Among them,one alkaline shock protein,asp23,was up-regulated most in the gentamycin-resistant strain（21.9-fold increase compared with the parental strain）.The result was further confirmed in a parallel reaction monitoring（PRM）experiment.（3）To validate the biological function of the asp23 gene,a gene disruption mutant was created.The asp23 gene disruption mutant（Zhang-G-1200-Δ0227）was significantly more sensitive to gentamycin compared with the evolved strain（Zhang-G-1200）,suggesting an important role of this gene in developing the gentamycin-resistant phenotype in Zhang-G-1200.There may be other multi-gene co-regulated so that the resistance of the mutant（MIC=8μg/mL）is still higher than that of the parent strain（MIC=2μg/mL）.Proteomics is a powerful tool to decode the molecular mechanism of adaptive evolution of probiotics.In this study,the adaptive evolutionary mechanism of probiotics under antibiotic-stress was explored on the proteomic level,and it lays a foundation for screening strong stress-resistant strains by metabolic engineering strategies in the future.