中国农业科学 ›› 2022, Vol. 55 ›› Issue (18): 3675-3684.doi: 10.3864/j.issn.0578-1752.2022.18.016

• 畜牧·兽医 • 上一篇    

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

贾雅婷(),胡慧慧,翟亚军,赵冰,何坤,潘玉善,胡功政,苑丽   

  1. 河南农业大学动物医学院,郑州 450046
  • 收稿日期:2021-08-02 接受日期:2021-10-27 出版日期:2022-09-16 发布日期:2022-09-22
  • 通讯作者: 苑丽
  • 作者简介:贾雅婷,Tel:13849102821;E-mail: 1218460153@qq.com
  • 基金资助:
    国家自然科学基金面上项目(31772800)

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

YaTing JIA(),HuiHui HU,YaJun ZHAI,Bing ZHAO,Kun HE,YuShan PAN,GongZheng HU,Li YUAN   

  1. College of Animal Medicine, Henan Agricultural University, Zhengzhou 450046
  • Received:2021-08-02 Accepted:2021-10-27 Online:2022-09-16 Published:2022-09-22
  • Contact: YUAN Li

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(traMtraJtraY)的mRNA表达量;构建LacZ融合报告菌株F25922/PM(PJ/PY)、FΔhns/PM(PJ/PY)和FΔhns/phns/PM(PJ/PY),分别测定不同菌株中3种基因(traMtraJtraY)启动子PM、PJ和PY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1 279.33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(traMtraJtraY)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1 510.14倍,其次为traYtraM,表达量分别为F25922的448.14倍和81.54倍,而回补株FΔhns/phns中不同基因的mRNA表达量均极显著低于FΔhns,与对照菌相类似。β-半乳糖苷酶活性测定结果显示,缺失报告菌株FΔhns/PM(PJ/PY)中PM、PJ和PY的β-半乳糖苷酶活性分别为5.66,10.45和21.91,均极显著高于对照菌F25922/PM(PJ/PY)的相应启动子(P<0.001),而回补报告菌株FΔhns/phns/PM(PJ/PY)中PM、PJ和PY的β-半乳糖苷酶活性极显著低于FΔhns/PM(PJ/PY),且均与对照菌株无明显差异。这些结果均表明hns对IncFⅡ质粒接合转移呈明显的负调控作用。EMSA结果显示,H-NS核蛋白均能明显阻滞3个tra基因启动子DNA的迁移,说明H-NS可直接与tra基因的3个启动子区结合;通过对结合位点的预测和EMSA进一步验证,证实H-NS核蛋白可与3个启动子区的AT富集区结合。【结论】H-NS核蛋白通过直接与IncFⅡ质粒的traMtraJtraY的启动子区的AT富集区结合,抑制启动子活性,从而导致启动子下游相关转移基因traMtraJtraY的表达量下降,结果明显抑制IncFⅡ质粒的接合转移。

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

Abstract:

【Objective】The objective of this study was to investigate the molecular mechanism of H-NS regulating conjugation of the IncFⅡ plasmid from a clinically isolated multi-drug resistant Escherichia coli from chicken, so as to provide a theoretical basis for controlling the rapid spread of IncFⅡ plasmid mediated multidrug resistance genes.【Method】The growth curves of Escherichia coli ATCC25922 and four recombinant strains (F25922, pBAD25922, 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 mRNA expression levels of IncFⅡ plasmid conjugation transfer related genes (traM, traJ and traY) in each recombinant strains (F25922, FΔhns and FΔhns/phns) were detected by RT-qPCR. The LacZ reporter strains F25922/PM(PJ/PY), FΔhns/PM(PJ/PY) and FΔhns/phns/PM(PJ/PY) were constructed to determine the β-galactosidase activity of three promoters of tra genes (traM, traJ and traY). 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 IncFⅡ plasmid 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 pBAD25922 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 IncFⅡ plasmid in FΔhns was 1 279.33 times higher than that of the control strain F25922 (P<0.001), and the FΔhns/phns conjugation frequency of the supplementary strain was significantly lower than that of FΔhns, although it did not completely recover to the level of the control strain F25922. Similarly, the mRNA expression levels of these tra genes (traM, traJ and traY) were significantly higher in the deletion mutant FΔhns. The mRNA expression level of traJ was the highest in FΔhns, which was 1 510.14 times that of F25922, followed by traY and traM, which were 448.14 times and 81.54 times that of F25922, respectively. Compared to the deletion strain FΔhns, expression levels of the tra genes (traM, traJ and traY) in the complemented strain FΔhns/phns were significantly decreased. The β-galactosidase activities of promoters PM, PJ and PY in the reporter strains FΔhns/PM (PJ/PY) were 5.66, 10.45 and 21.91, respectively, which were significantly higher than that of the corresponding promoters of F25922/PM (PJ/PY) (P<0.001). The activities of promoters PM, PJ and PY in the complement reporter strains FΔhns/phns/PM (PJ/PY) were significantly lower than that of FΔhns/PM (PJ/PY), and there was no significant difference with the control strain F25922/PM (PJ/PY). EMSA results showed that H-NS protein could block the DNA migration of three promoters of tra genes, indicating that H-NS could directly bind to the three promoters. By predicting the binding sites and further verified by EMSA, it was confirmed that H-NS protein could bind directly to the AT enrichment region of the promoters of the three genes (traM, traJ and traY).【Conclusion】H-NS protein could bind directly to the AT enrichment region of the promoter region of IncFⅡ plasmid conjugation transfer related genes (traM, traJ and traY), and negatively regulate the conjugation transfer of IncFⅡ plasmid by inhibiting the activity of promoter.

Key words: H-NS, tra gene, IncFⅡ, plasmid mating, negative regulation, E. coli from chicken