Regulatory Mechanism Of H-NS And CpxR On Tra Genes Of IncFⅡ Plasmid

The horizontal spread of plasmid-mediated drug resistance genes is one of the important reasons for the rapid increase of multiple drug-resistant bacteria in clinic.IncFII plasmid is the common conjugate plasmid carrying blacTX-M gene in Escherichia coli,which can be horizontally transmitted among different genera.H-NS nucleoprotein is a common negative regulatory protein,which can regulate the transcription and expression of exogenous genes.CpxR is a regulatory protein in the two-component signal transduction system CpxAR,which can regulate the transcription and expression of multiple genes in bacteria.Our laboratory has previously proved that H-NS and CpxR could negatively regulate the conjugation of IncFII plasmid.However,the specific regulation mechanism(direct or indirect regulation)and regulation sites are not clear.In this study,the plasmid IncFII of clinically isolated multidrug-resistant Escherichia coli was used as the research object.The H-NS and CpxR prokaryotic expression vectors were constructed using pET-21b as vector,and purified H-NS and CpxR proteins were obtained by nickel ion affinity chromatography.The conjugation related genes tra(traM,traJ and traY)of IncFII plasmid were amplified by PCR.EMSA showed that both H-NS and CpxR proteins could significantly block the migration of the promoter DNA of the three tra genes,indicating that H-NS and CpxR could regulate the conjugation of the IncFII plasmid by directly binding to the three promoters of the tra gene.The binding sites of H-NS and CpxR to the tra gene promoters were further identified by EMSA by gradually shortening the length of the tra genes promoter regions,and the regulatory sites were verified by DNase I footprint test.Finally,the binding sites of H-NS to the promoter regions of traM,traJ and traY genes were determined to be PM25:5’-TTGTTTCAGAATATAAAAGGT ACTG-3’;PJ25:5’-ATTGAAACTGAAAATCGCCGATGCA-3’ and PY20:5’-GTTAAGTAAATGTTAAATAA-3’,respectively.The binding sites of CpxR to the promoter regions of traM,traJ and traY genes are 5’-TTTACATT-3’(PM);5’一ATAAGAAT-3’(PJ)and 5’-AATTTTAT-3’(PY),respectively.Three potential CpxR boxes were identified as 5’-TTACA-N3-TTA AA-3’(PM),5’-TCAAA-N4-AGAAT-3’(PJ)and 5’-TTTCT-N13-TTTAT-3’(PY),respectively.Three 24 bp DNA sequences(PM24;PJ24 and PY24)covering CpxR binding sites were designed for EMSA test with H-NS,respectively.The results showed that H-NS could bind to CpxR binding sites,which proved that H-NS and CpxR could antagonize each other by competing for the same binding sites.The mRNA expression levels of hns and cpxR in the recombinant strains(F25922,FΔcpxR,FΔcpxR/FΔhns,FΔhns,FΔhns/phns,FΔcpxRΔhns FΔcpxRΔhns/pcpxR and FΔcpxRΔhns/phns)were determined by RT-PCR.The results showed that the expression levels of hns and cpxR genes in the deletion strain were significantly decreased,while the expression levels of hns and cpxR genes in the replacement strain were increased,but did not recover to the level of the control strain F25922.This explained the reason why the expression levels of tra genes did not recover to the original level in the replacement plants determined earlier.In conclusion,this study identified the regulatory sites of H-NS and CpxR on the IncFII plasmids tra gene promoters,providing a theoretical basis for clinical in-depth study on the control of IncFII plasmids mediated rapid horizontal spread of multiple drug resistance genes.