Research On The Function Of QS System And Rmf Gene Of E.coli Under Stress Condition

Enterohemorrhagic Escherichia coli O157:H7 is a kind of foodborne pathogen,which often colonizes in the gastrointestinal tract of the host and causes diseases,such as hemorrhagic colitis and hemolytic uremic syndrome,which seriously endanger human life and health.When such pathogenic Escherichia coli enters into the gastrointestinal tract,the acidic environment in the host stomach is a natural barrier.EHEC O157:H7 has strong acid resistance.By activating a complex acid resistance regulation network,it can withstand acidic conditions with pH 2.5 or lower for several hours to enter into the intestine through the acidic environment.Quorum sensing(QS)is an intercellular signal communication mechanism that depends on population density in bacteria.AHL(acyl homoserine lactones)is a signal molecule of bacterial type-? quorum sensing system.E.coli can not synthesize AHL by itself due to lack of LuxI,but encodes the transcription factor SdiA,the AHL receptor.Therefore,it can "quorum sense eavesdrop" the AHLs produced by other bacteria and respond on a population scale.Previous studies in our laboratory found that compared with other short chain AHLs(C4-HSL and C6-HSL),3-oxo-C6-HSL with specific group at 3-carbon position improved the acid tolerance of EHEC EDL933 more significantly.This study aims to investigate the mechanism of type-I quorum sensing system regulating E.coli acid resistance,and further clarify its mechanism.The specific research content is as follows:1.Screening the main differentially expressed genes regulating EHEC acid resistance by QS based on RNA-seq technologyThe previous laboratory study showed that 100 ?M AHL molecule 3-oxo-C6-HSL significantly improved the survival ability of EHEC O157:H7 under strong acid conditions.Therefore,100?M 3-oxo-C6-HSL was used in this study to stimulate O157:H7 EDL933 strain.RNA-seq technology was used to screen the differentially expressed genes of bacteria stimulated by quorum sensing under pH 2.5 acidic conditions.Transcriptome results showed that 36 genes were significantly up-regulated and 47 genes were significantly down regulated in AHL group compared with the control group.GO enrichment analysis and KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in the ribosomal pathway,in which the ribosome modulation factor coding gene rmf and ribosome-associated inhibitor A coding gene raiA were up-regulated,and the ribosomal protein related coding genes such as rpsL,rpsT,rpsJ,rplM and rplY were down regulated.Both rmf and raiA played important roles in ribosome hibernation,but rmf had a wider effect,and with the most upregulation multiples(2.9 multiples).Therefore,rmf gene was selected as the target gene for subsequent research.This study is expected to lay the foundation for further exploring the acid tolerance mechanism of O157:H7 EDL933 strain regulated by quorum sensing molecules.2.Construction of Escherichia coli ?rmf strain and verification of rmf regulating multiple stress responses of Escherichia coliAmong the differentially expressed genes,the up-regulation multiple of ribosome modulation factor rmf was most remarkable.Therefore,rmf gene was selected as the target gene for subsequent study.Under stress conditions,RMF binds two 70S ribosomes and changes the conformation of 30S subunit to promote ribosome dimerization and form inactive 100S ribosomes,which is an important survival strategy for bacteria.In this study,we constructed two rmf mutant strains EHEC EDL933 ?rmf and APEC TW-XM ?rmf,two complemental strains EHEC EDL933 ?rmf/prmf and APEC TW-XM ?rmf/prmf,then explored the regulatory role of RMF in acid stress,cold stress,heat stress,starvation stress and oxidative stress.The results showed that after starvation treatment,compared to the wild type strain,the survival rate of EDL933 ?rmf decreased by 13.7 times,and TW-XM ?rmf strain decreased by 5.2 times.After cold treatment(-20?),compared to the wild type strain,the survival rate of EDL933 ?rmf decreased by 8.7 times,and TW-XM ?rmf decreased by 5.7 times.After heat treatment(50?),compared to the wild type strain,the survival rate of EDL933 ?rmf decreased by 7.0 times,and TW-XM ?rmf decreased by 5.0 times.5 mmol/L H2O2 was added as the oxidative stress condition to measure the growth curve.Compared to the wild type strain,the growth of EDL933 ?rmf and TW-XM ?rmf were more inhibited,the OD600 of ?rmf strain was only 50%and 40%of that of EDL933 and TW-XM wild strains at the 9th hour.After acid treatment(pH 2.5),compared to the wild type strain,the survival rate of EDL933 ?rmf decreased by 9.0 times,and TW-XM ?rmf decreased by 5.0 times.In conclusion,RMF played an important role in the resistance to starvation stress,cold stress,heat stress,oxidative stress and acid stress for EHEC EDL933 and APEC TW-XM.3.EHEC mediated QS signal molecules AHLs and rmf gene to regulate acid toleranceAfter determining the up-regulation of rmf gene expression by quorum sensing signal molecule AHL,the mechanism of rmf gene participating in the regulation of EHEC acid resistance was further discussed.Firstly,the changes of acid tolerance in EHEC stimulated by exogenous and endogenous AHL were verified.The acid resistance experiment showed that exogenous addition of AHL could enhance the acid resistance of O157:H7 EDL933.Compared with 10 ?M 3-oxo-C6-HSL,the effect of 100 ?M 3-oxo-C6-HSL was more significant.The yenI gene of Yersinia enterocolitica was introduced into the EDL933,which offered the ability to synthesize short-chain AHL.The acid resistance experiment showed that endogenous AHL could enhance the acid response ability of EDL933.The wild type strain,?rmf strain and ?rmf/prmf strain were cultured to the stable growth stage,and the acid resistance experiment showed that the survival rate of ?rmf strain was 8 times lower than that of wild type strain under pH 2.5 conditions,and the acid resistance of ?rmf/prmf strain was restored.This is the first time that rmf gene was found to be involved in the acid resistance process of O157:H7 EDL933.The results of fluorescence quantitative PCR showed that RMF enhanced the acid tolerance of EHEC EDL933 by regulating the expression of glutamate acid tolerance system genes(gadA?gadC?gadW?gadE),arginine acid tolerance system gene(adiA)and molecular chaperones(hdeA?hdeB).In addition,AHL regulated the expression of rmf gene via SdiA receptor pathway,and the regulatory effect was only activated under acid stress conditions;rmf had significantly regulation effect on rpoS and sdiA gene under acid stress conditions.Therefore,there existed mutual regulation effects among sdiA?rpoS and rmf,which was also one of the pathways by which rmf participated in the regulation of E.coli acid tolerance.In conclusion,QS signal molecules AHLs regulated the expression of a variety of acid tolerance related genes by regulating ribosome modulation factor rmf to enhance its acid resistance,and rmf gene also affected the viability of E.coli under a variety of stress conditions.The results of this study laid a foundation for further improving the stress response mechanism of E.coli.