河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇中vgrG操纵子的调控研究

韩雨 黄元铭 程倩 李杰 刁保卫 梁未丽

韩雨, 黄元铭, 程倩, 李杰, 刁保卫, 梁未丽. 河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇中vgrG操纵子的调控研究[J]. 疾病监测, 2022, 37(5): 585-590. doi: 10.3784/jbjc.202203180100
引用本文: 韩雨, 黄元铭, 程倩, 李杰, 刁保卫, 梁未丽. 河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇中vgrG操纵子的调控研究[J]. 疾病监测, 2022, 37(5): 585-590. doi: 10.3784/jbjc.202203180100
Han Yu, Huang Yuanming, Cheng Qian, Li Jie, Diao Baowei, Liang Weili. Regulation of VfqI-VfqR Quorum sensing circuit on vgrG operon in VflT6SS orphan cluster of Vibrio fluvialis[J]. Disease Surveillance, 2022, 37(5): 585-590. doi: 10.3784/jbjc.202203180100
Citation: Han Yu, Huang Yuanming, Cheng Qian, Li Jie, Diao Baowei, Liang Weili. Regulation of VfqI-VfqR Quorum sensing circuit on vgrG operon in VflT6SS orphan cluster of Vibrio fluvialis[J]. Disease Surveillance, 2022, 37(5): 585-590. doi: 10.3784/jbjc.202203180100

河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇中vgrG操纵子的调控研究

doi: 10.3784/jbjc.202203180100
基金项目: 国家重点研发计划(No. 2021YFC2300302);国家自然科学基金(No. 81772242)
详细信息
    作者简介:

    韩雨,女,山东省枣庄市人,在读硕士研究生,主要从事河弧菌致病及调控研究,Email:405906260@qq.com

    通讯作者:

    梁未丽,Tel:010–58900742,Email:liangweili@icdc.cn

  • 中图分类号: R211; R378.2

Regulation of VfqI-VfqR Quorum sensing circuit on vgrG operon in VflT6SS orphan cluster of Vibrio fluvialis

Funds: This study was supported by the National Key Research and Development Program (No. 2021YFC2300302) and the National Natural Science Foundation of China (No. 81772242)
More Information
  • 摘要:   目的  探究河弧菌VfqI-VfqR密度感应系统对VflT6SS2附属基因簇hcp-vgrGvgrG操纵子的调控。   方法  使用荧光定量反转录聚合酶链式反应(qRT-PCR)检测野生株和VfqI-VfqR系统缺失株中vgrG的mRNA水平。 利用启动子-lux报告质粒系统检测冷光值,确定vgrG启动子在河弧菌野生株、缺失株、回补株中的活性差异。 在大肠埃希菌中,导入VfqR过表达质粒,检测vgrG启动子-lux报告质粒冷光值,确定VfqR可否激活vgrG启动子。  结果  ∆vfqI、∆vfqR和∆vfqI-vfqR缺失株中vgrG mRNA水平和启动子活性均显著低于野生株;在∆vfqR中回补VfqR表达质粒pSRvfqR和在∆vfqI中分别添加3-oxo-C10-HSL、C10-HSL和3-oxo-C12-HSL AHLs分子均能恢复vgrG启动子活性,并且3种AHLs分子效果大体一致。 在大肠埃希菌中过表达VfqR并添加上述3种外源AHLs,对vgrG启动子活性没有影响。  结论  VfqI-VfqR密度感应系统在启动子水平正性调控vgrG操纵子,并且该调控作用是间接的。
  • 图  1  VfqI-VfqR 密度感应系统基因组织结构和序列特征

    Figure  1.  Structure and sequence feature of VfqI-VfqR quorum sensing circuit

    图  2  野生株和VfqI-VfqR 密度感应系统缺失株中vgrG的mRNA水平

    注:a. P<0.05

    Figure  2.  mRNA levels of vgrG in wide V. fluvialis strain and VfqI-VfqR QS deletion mutant of V. fluvialis

    图  3  野生株和VfqI-VfqR 密度感应系统缺失株中vgrG启动子活性

    注:a. P<0.05;85003与ΔvfqR在4 h和7 h时,t=10.44和6.32,均P<0.05;85003与ΔvfqI在4 h和7 h时,t=11.67和15.01,均P<0.05;85003与ΔvfqI-vfqR在3 h和5 h时,t=7.867和16.82,均P<0.05

    Figure  3.  Promoter activities of vgrG in wild V. fluvialis strain and VfqI-VfqR QS deletion mutant of V. fluvialis

    图  4  添加外源AHLs分子对ΔvfqIvgrG启动子活性的影响

    注:a. P<0.05;未添加AHLs组中WT与ΔvfqIt=11.23,P<0.05;ΔvfqI与ΔvfqI添加5 μmol/L 和25 μmol/L 3-oxo-C10-HSL组,t=41.45和31.89,P<0.05;ΔvfqI与ΔvfqI添加5 μmol/L 和25 μmol/L C10-HSL组,t=16.57和19.65,P<0.05;ΔvfqI与ΔvfqI添加5 μmol/L 和25 μmol/L 3-oxo-C12-HSL组,t=22.63和3.69,P<0.05;ΔvfqI与ΔvfqI添加5% AHLs组,t=43.51,P<0.05

    Figure  4.  Effects of adding exogenous AHLs on vgrG promoter activity in ΔvfqI

    图  5  ΔvfqR中回补pSRvfqRvgrG启动子活性的影响

    注: a. P<0.05;WT. 河弧菌85003;3 h时,pSRKTc/WT与pSRKTc/ΔvfqRt=6.363,P<0.05;pSRKTc/ΔvfqI与pSRvfqRvfqRt=8.750,P<0.05。7 h时,pSRKTc/WT与pSRKTc/ΔvfqRt=23.55,P<0.05;pSRKTc/ΔvfqI与pSRvfqRvfqRt=19.72,P<0.05

    Figure  5.  Influence of pSRvfqR complementation on vgrG promoter activity in ΔvfqR

    图  6  大肠埃希菌中VfqR对vgrG启动子的调控

    Figure  6.  Regulation of VfqR on vgrG promoter activity in E. coli

    表  1  本实验中使用的菌株及质粒

    Table  1.   Strains and plasmids used in this study

      菌株/质粒相关特性来源
    大肠埃希菌
     SM10λpirthr thi tonA leu supE lacY recA::RP4-2Tc::Mu (λpirR6K), KmR实验室保存
     pSRKTc/ SM10λpir含有空载质粒pSRKTc的 SM10λpir,KmR,TetR实验室保存
     pSRvfqR/ SM10λpir含有VfqR过表达质粒pSRvfqR的 SM10λpir,KmR,TetR实验室保存
     ptssI2a-lux/ SM10λpir含有vgrG启动子-lux融合报告质粒ptssI2a-lux的SM10λpir,KmR,CmR实验室保存
     pSRKTc ptssI2a-lux / SM10λpir含有空载质粒pSRKTc和vgrG启动子-lux融合报告质粒ptssI2a-lux的 SM10λpir,KmR,TetR,CmR本研究构建
     pSRvfqR+ ptssI2a-lux/ SM10λpir含有VfqR过表达质粒pSRvfqRvgrG启动子-lux融合报告质粒ptssI2a-lux的 SM10λpir,KmR,TetR,CmR本研究构建
    河弧菌
     85003河弧菌,野生株,SmR实验室保存
     ΔvfqR河弧菌85003 vfqR缺失株,SmR20
     ΔvfqI河弧菌85003 vfqI缺失株,SmR20
     ΔvfqR-vfqI河弧菌85003 vfqRvfqI双缺失株,SmR实验室保存
     ptssI2a-lux/WT含有冷光报告质粒ptssI2a-lux的野生株,SmR ,CmR15
     ptssI2a-luxvfqR含有冷光报告质粒ptssI2a-lux的ΔvfqR,SmR , CmR本研究构建
     ptssI2a-luxvfqI含有冷光报告质粒ptssI2a-lux的ΔvfqI,SmR , CmR本研究构建
     ptssI2a-luxvfqR-vfqI含有冷光报告质粒ptssI2a-lux的ΔvfqR-vfqI,SmR , CmR本研究构建
     pSRKTc+ ptssI2a-lux/WT含有pSRKTc空载和ptssI2a-lux的野生株,TetR,SmR , CmR本研究构建
     pSRKTc+ ptssI2a-luxvfqR含有pSRKTc空载和ptssI2a-luxvfqR缺失株,TetR,SmR , CmR本研究构建
     pSRvfqR+ ptssI2a-luxvfqR含有pSRvfqR和ptssI2a-luxvfqR缺失株,TetR,SmR , CmR本研究构建
    质粒
     pSRKTc表达载体质粒,lac promoter, lacIqlacZα,TetR实验室保存
     ptssI2a-lux含有VflT6SS2 vgrG-A启动子区域的pBBRlux,CmR实验室保存
     pSRvfqR含有vfqR ORF的pSRKTc, TetR实验室保存
    注:Km. 卡那霉素,Sm. 链霉素,Cm. 氯霉素,Tet. 四环素
    下载: 导出CSV
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  • 收稿日期:  2022-03-18
  • 网络出版日期:  2022-04-13
  • 刊出日期:  2022-05-31

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