The Regulatory Mechanism On The Expression Of Antibiotic Resistance Gene

It is well-known that the aggravating antimicrobial resistance in bacteria has become one of the biggest threat to human’s health.The abuse of antibiotics is the primary reason for this problem.Antibiotics residues in the environment can not only form selective pressure,endowing the survival advantage of drug-resistant bacteria and helping the transfering of antibiotic resistance genes(ARGs)between homologous and heterogeneous bacteria,but also regulate mobile genetic elements such as antimicrobial resistance gene-carrying integrons and lead to the spread of antimicrobial resistance genes.The typical class 1 integron structure includes 5’conserved segment(5’ CS),3’conserved segement(3’CS)and Variable region(VR).Class 1 integron is the most common among all the integrons,it allows bacteria to capture or,exchange as well as express gene cassettes.The integron 5’CS is composed of a gene intI,which encodes a site-specific recombinase IntI,and a recombination site attI,the gene cassette promoter P,with divergent to the integrase gene and a Pint promoter which regulates the transcription of integrase.Pint is located outside of the intI coding regions.By sequencing the experimental strains,we found that our strains P,is located within the intI coding regions,but,Pint is not.In addition,there is a repeated sequence between the divergent Pc and Pnt.This results can provide a certain basis for the co-regulation of IntI and gene cassette.Indeed,some studies have shown that the expression of integrase and gene cassette is co-regulated in V.cholera.According the difference of the predicted-10 region and the predicted-35 region of Pc promoter,there are different types of Pc,Based on our previous findings we found that the most abundant Pc promoters in Ji’nan’s water environment are PcW、PcWTGN-10,PcH10.There is a cAMP-CRP box upstream of Pc promoter which the key regulatory proteins of the cAMP-CRP pathway CRP can combined with,and a LexA box upstream of the Pint which the key regulatory proteins of SOS response LexA can combined with.According this we can conclude that SOS response or cAMP-CRP pathway can regulate the integrase or gene cassette.SOS response or cAMP-CRP pathway can also respond to environmental pressures,and for bacteria,the residual antibiotics in the environment are a kind of pressure.Our studies also found that the cAMP-CRP pathway can regulate the integrase expression.However,does antibiotic residues in the environment and SOS or cAMP-CRP pathway affect the expression of gene cassettes?In this project,we aim to further our understanding on the impact of antibiotics from the perspective of how antibiotics regulate the expression of antimicrobial resistance genes in integrons.On the basis of previous studies,we first knockout the crp,then we constructed three bidirectional promoter vectors with the the P,variants and Pint,observing the changes of P,and Pint when they were induced by different conditions and analyzing their regulatory factors.After the induction of different antibiotics,on the one hand,we detect the transcriptional level of CRP by reverse transcription quantitative real-time PCR(RT-qPCR),on the other hand we use RT-qPCR to detect the reporter gene of the P.and Pint.In our studies we found that antibiotics can induce the expression of antimicrobial resistance genes in integrons,and antibiotics can induce the cAMP-CRP pathway too.After the induction of crp positive bacteria and crp negative bacteria,we found that antimicrobial resistance genes expressions in different degree,the former is higher than the latter,so we can conclude that cAMP-CRP pathway can regulate the expression of antimicrobial resistance genes in integrons.Residual antibiotics in the environment can not only regulates the expression of integrase,but also regulates the expression of gene cassettes,which will all help bacteria to increase their drug resistance.This work will further our understanding on the mechanism by which antibiotics regulate antimicrobial resistance,and provide the theoretical basis on the control of antimicrobial resistance.