The Oxidative Stress Response Mechanism And Functional Role Of Transcription Factor CopR In Oxidative Stress In Lactobacillus Plantarum CAUH2

Lactobacillus plantarum are natural inhabitants of the human intestinal tract.Some strains in this species are well known for their health-promoting effects and used as probiotics in fermented dairy production.In the process of industrial production and storage,however,oxidative stress can cause oxidative damage of proteins and DNA and other biological molecules and further affect the survival rate of L.plantarum in fermented foods.In this study,RNA-Seq transcriptomic analysis was employed to investigate the cellular response to oxidative stress in L.plantarum CAUH2,and functional studies of key transcription factor in oxidative stress were performed by molecular biology methods.The results will provide more valuable information for comprehensively understanding the mechanism of oxidative stress response in L.plantarum.The main contents and results of this dissertation are as follows.(1)Construction of the transcriptomic profiles of L.plantarum CAUH2 under oxidative stress conditions.The complete genome sequencing of L.plantarum CAUH2 was performed by Illumina Hiseq2000/Miseq platform.A total of 353 MB of clean data was obtained with a sequencing depth of 108x.After annoting by PGAP and analysis by COG cluster,the results were used as the reference for further transcriptome research.Early-exponential-phase CAUH2 cells(OD600nm=0.8)were treated with 5 mM H2O2 for 30 min.The Next-Generation Sequencing(NGS)based RNA-Seq revealed that the transcript levels of 306 genes were significantly changed(|log2 fold_change|>3,p<0.001),including 189 genes up-and 117 genes down-regulated.These results demonstrated that the bile oxidative response in L.plantarum CAUH2 was a complex physiological regulation network.(2)Bioinformatics analysis of the mechanisms of oxidative stress response in L.plantarum CAUH2.The oxidative stress response mechanisms were predicted on the basis of functional annotation of differentially expressed genes according to UniPort,KEGG and NCBI database.The results indicated that the EMP pathway was accelerated by multiple adjustments including up-regulating enzyme expression,increasing substrate supply and reducing product accumulation to generate more energy to cope with oxygen stress;pentose phosphate pathway was enhanced to accelerate the generation of intracellular reducing power NADPH;strengthen the control of intracellular oxygen reduction and increase the activity of peroxidases and protein repair enzymes by controlling the activity of the thioredoxin system;regulate the homeostasis of transition metal ions such as Fe and Cu to prevent the occurrence of Fenton reaction and reduce the generation of reactive oxygen species.Furthermore,mdxF mutant was constructed by insertional inactivation method due to its most significant changes at transcriptional levels.H2O2 challenge assays indicated a significantly higher sensitivity of mdxF mutants,indicating the important antioxidant role of MdxF in L.plantarum CAUH2.(3)The regulatory mechanisms of transcription factor CopR in response to oxidative stress.41 Homologous over-expression strains of transcription factors were constructed in L.plantarum CAUH2 using the constitutive expression vector pSlpA8148.H2O2 challenge of over-expression strains indicated three functional transcription factors,TreR1,MntR and CopR.The antioxidant functions of CopR were further confirmed by the increased survival of its insertional mutant when exposed to H2O2.Through genetic structure analysis and search for the DNA-binding sites "cop box",we predicted that gene copB encoding a copper transporting ATPase was the target gene of CopR,and the DNA-binding specificity of CopR to target gene copB was assessed by EMSA in vitro.Mutation of copB lead to increased sensitivity to oxidative and copper stress.and EMSA analysis showed that the binding intensity of CopR with the probes became weak with an increasing concentration of copper,suggesting that the CopR can be released from the cop box in the promoter of CopB by Cu2+.These results revealed that CopR functions as a regulator that negatively regulates copB gene and Cu2+ serves as inducer of CopR to activate the ATPase CopB to export cupric ions under H2O2 stress in L.plantarum CAUH2.In summary,this study represented the first RNA-seq based transcriptomic of oxidative stress response in L.plantarum.The results will provide new insights into the mechanisms that enable L.plantarum CAUH2 to cope with oxidative stress.