The Coordinate Regulation Mechanism Of H-NS And CpxAR On IncF? Plasmid Conjugation

Plasmid mediated horizontal spread of drug resistance genes is the most common way to cause the spread of drug resistance genes in bacteria.IncF?plasmid is one of the common conjugation plasmids in multidrug-resistant E.coli.Its conjugation is regulated by a variety of factors,among which the regulation of systemic/local regulatory proteins(such as H-NS,CpxAR and Tra)is particularly important.However,the molecular mechanism by which H-NS and CpxAR interact to regulate IncF? plasmid conjugation remains unclear.A series of cpxR and hns deletion mutants and the complement strains of the mutants were constructed by Red homologous recombination.The IncF? plasmids carrying blaCTX-M-65,fosA3 and rmtB resistance genes were electrically transferred into the above constructed strains,and 8 recombinant strains containing IncF? plasmids were obtained.Firstly,the test was carried out:the antimicrobial sensitivity,growth curve,plasmid stability of each strain were determined by microbroth dilution method and the measurem of the copy number of the pBAD plasmid and IncF? plasmid.Secondly,J53 was used as the receptor bacterium to perform the plasmid mating test and calculate the conjugation frequency and analyze the characteristics of the plasmid conjugation of the recombinant bacteria.At the same time,the relative mRNA expression levels of tra,conjugation and transfer inhibitors fino,active efflux pump gene arcA,and outer membrane protein ompA in recombinant bacteria were detected by fluorescence quantitative PCR.Finally,promoter regions that may be regulated were connected to the upstream of the report plasmid LacZ gene to construct reported strains,and the effect of CpxAR and H-NS on the activity of PY,PJ and PM promoters was analyzed by the activity of ?-galactosidase.Phenotypic results showed that after cpxR was absent,the sensitivity to gentamicin was increased,while the sensitivity to ampicillin,doxycycline and florfenicol was decreased.After the absence of hns,the sensitivity to gentamicin,amikacin,doxycycline and flurbenicol increased,while the sensitivity to ampicillin decreased.In addition,the deletion of cpxR gene had no significant effect on the growth rate of bacteria,but the deletion of hns gene led to the poor growth adaptability of bacteria and the slow growth rate of bacteria.Plasmid pEC011 stability is poor in the double-deletion mutants F?cpxR?hns.The results of conjugation frequency showed that cpxR and hns had a negative regulatory effect on conjugation transfer of IncF? plasmid.H-NS and CpxAR might antagonistically regulate plasmid pEC011(IncF?)mating.Relative mRNA expression results showed that the hns of E.coli significantly down-regulated the mRNA expression levels of traJ,traY,traM,traL,traF,traE and traA,and had stronger regulatory effects on traJ and traE.CpxR significantly down-regulated the mRNA expression of traY,traA,traJ and traM,and up-regulated the mRNA expression of traL,traE and fino,with no significant regulatory effect on traF,ompA and acrA.It was further demonstrated that CpxR and H-NS play an antagonistic role in regulating tra related genes.The activity of?-galactosidase indicated that cpxR could reduce the activity of tra related promoters PY,PJ and PM.hns also attenuated the activity of tra related promoters PY,PJ and PM.And the regulation of PY promoter is stronger.CpxR and H-NS had an antagonistic effect on the regulation of the promoter activities of PY,PJ and PM.In summary,nuclear protein H-NS and two-component signal transduction system CpxAR have an antagonistic effect on the conjugation transfer,which provides a theoretical basis for the control of IncF? plasmid mediated rapid spread of multi-drug resistance genes.