核蛋白H-NS调控多重耐药鸡大肠埃希菌IncFⅡ质粒接合转移的分子机制  

Molecular Mechanism of Regulation by H-NS on Inc FⅡ Plasmid Transmission of Multi-drug Resistant Chicken Escherichia coli

作　　者：贾雅婷 胡慧慧 翟亚军 赵冰 何坤 潘玉善[1] 胡功政[1] 苑丽[1] JIA YaTing;HU HuiHui;ZHAI YaJun;ZHAO Bing;HE Kun;PAN YuShan;HU GongZheng;YUAN Li(College of Animal Medicine,Henan Agricultural University,Zhengzhou 450046)

机构地区：[1]河南农业大学动物医学院,郑州450046

出　　处：《中国农业科学》2022年第18期3675-3684,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31772800)。

摘　　要：【目的】以临床分离的多重耐药鸡大肠埃希菌IncFⅡ质粒为研究对象,探究核蛋白H-NS调控其接合转移的分子机制,为控制IncFⅡ质粒介导的多重耐药基因水平散播提供理论依据。【方法】测定大肠埃希菌ATCC25922及4株重组菌(pBAD25922、F25922、FΔhns和FΔhns/phns)的生长曲线,比较hns对菌株的影响情况;分别以F25922、FΔhns和FΔhns/phns为供体菌,大肠埃希菌J53(耐叠氮钠)为受体菌,进行接合试验,并计算接合频率;采用实时荧光定量PCR检测各重组菌(F25922、FΔhns和FΔhns/phns)中IncFⅡ质粒接合转移相关基因(traM、traJ和traY)的mRNA表达量;构建LacZ融合报告菌株F25922/P_(M)(P_(J)/P_(Y))、FΔhns/P_(M)(P_(J)/P_(Y))和FΔhns/phns/P_(M)(P_(J)/P_(Y)),分别测定不同菌株中3种基因(traM、traJ和traY)启动子P_(M)、P_(J)和P_(Y)的β-半乳糖苷酶活性;构建H-NS蛋白原核表达载体,采用镍离子亲和层析法分离纯化H-NS核蛋白,PCR扩增并纯化3种基因启动子区的DNA序列,利用电泳迁移率变动分析试验(EMSA)探明核蛋白H-NS调控IncFⅡ质粒接合作用的调控方式,预测H-NS与不同启动子的结合位点并用EMSA进行进一步验证。【结果】重组菌F25922和pBAD25922与大肠埃希菌ATCC25922的生长状况无明显差异,说明质粒pBAD和IncFⅡ均不影响宿主菌大肠埃希菌ATCC25922的生长;但是,缺失重组菌FΔhns和缺失回补重组菌FΔhns/phns的生长速度明显低于大肠埃希菌ATCC25922,表明hns的缺失导致菌株的生长适应性变差,但不影响菌株的存活。接合试验结果显示,FΔhns中IncFⅡ质粒的接合频率较对照菌F25922升高了1279.33倍(P<0.001),而FΔhns/phns中IncFⅡ质粒的接合频率虽未完全恢复到对照菌株的水平,但也明显低于FΔhns。实时荧光定量PCR结果显示,FΔhns中tra基因(traM、traJ和traY)的mRNA表达量均极显著高于对照菌株(P<0.001),其中traJ的mRNA表达量最高,为F25922的1510.14倍,其次为traY和traM,表达量�【Objective】The objective of this study was to investigate the molecular mechanism of H-NS regulating conjugation of the Inc FⅡplasmid from a clinically isolated multi-drug resistant Escherichia coli from chicken,so as to provide a theoretical basis for controlling the rapid spread of Inc FⅡplasmid mediated multidrug resistance genes.【Method】The growth curves of Escherichia coli ATCC25922 and four recombinant strains (F25922,p BAD25922,FΔhns and FΔhns/phns) were determined to compare the influence of hns on different strains.The conjugation experiments were conducted with F25922,FΔhns and FΔhns/phns as donors and Escherichia coli J53 as recipient,then the conjugation frequency was calculated.The m RNA expression levels of Inc FⅡplasmid conjugation transfer related genes (tra M,tra J and tra Y) in each recombinant strains (F25922,FΔhns and FΔhns/phns)were detected by RT-q PCR.The Lac Z reporter strains F25922/P(P/P),FΔhns/P(P/P) and FΔhns/phns/P(P/P) were constructed to determine the β-galactosidase activity of three promoters of tra genes (tra M,tra J and tra Y).The H-NS protein was purified by Ni-NTA resin affinity chromatography.The DNA sequences of three promoters of tra genes were amplified by PCR.The mechanism of H-NS regulating Inc FⅡplasmid transmission was identified by EMSA,and the binding sites of H-NS to different promoters were predicted and further verified by ESMA.【Result】The growth of recombinant strains F25922 and p BAD25922 were not significantly different from that of Escherichia coli ATCC25922,while the growth rate of deleted recombinant strain FΔhns and complemented strain FΔhns/phns were significantly lower than that of the control strain F25922.The results showed that the absence of hns could make the adaptability of strains worse,but did not affect the survival of the strains.The results of the conjugation test showed that the conjugation frequency of Inc FⅡplasmid in FΔhns was 1 279.33times higher than that of the control strain F25922 (P<0.001),and the FΔ

关 键 词：H-NS tra基因 IncFⅡ质粒 质粒接合作用 负调控 鸡大肠埃希菌 

分 类 号：S852.61[农业科学—基础兽医学]