Zhou Hang, Zheng Cao, Su Jianmei, Chen Bo, Fu Yang, Xie Yuqun, Tang Qing, Chou Shan-Ho, He Jin
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Wuhan, Hubei 430068, People's Republic of China.
Sci Rep. 2016 Feb 19;6:20871. doi: 10.1038/srep20871.
c-di-GMP riboswitches are structured RNAs located in the 5'-untranslated regions (5'-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5'-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results showed that this natural triple-tandem riboswitch controlled the expression of the reporter gene more stringently and digitally than the double-tandem or single riboswitch. A sandwich-like dual-fluorescence reporter was further constructed by fusing the Bc3-5 RNA gene between the two fluorescence protein genes amcyan and turborfp. This reporter strain was found to exhibit detectable fluorescence color changes under bright field in response to intracellular c-di-GMP level altered by induced expression of diguanylate cyclase (DGC) PleD. Using this system, two putative membrane-bound DGCs from B. thuringiensis and Xanthomonas oryzae were verified to be functional by replacing pleD with the corresponding DGC genes. This report represented the first native triple-tandem riboswitch that was applied to serve as a riboswitch-based dual-fluorescence reporter for the efficient and convenient verification of putative DGC activity in vivo.
环二鸟苷酸核糖开关是位于信使核糖核酸(mRNA)5'-非翻译区(5'-UTR)的结构化核糖核酸,可根据第二信使环二鸟苷酸浓度的变化来调节下游基因的表达。我们发现了三个结构相似的完整环二鸟苷酸核糖开关(Bc3、Bc4和Bc5 RNA),它们串联排列,在苏云金芽孢杆菌亚种中国CT-43的cspABCDE mRNA的5'-UTR中构成一个三串联(Bc3-5 RNA)核糖开关。我们的结果表明,这种天然的三串联核糖开关比双串联或单核糖开关更严格、更数字化地控制报告基因的表达。通过将Bc3-5 RNA基因融合在两个荧光蛋白基因amcyan和turborfp之间,进一步构建了一种三明治样双荧光报告基因。发现该报告菌株在明场下可响应由双鸟苷酸环化酶(DGC)PleD的诱导表达而改变的细胞内环二鸟苷酸水平,表现出可检测的荧光颜色变化。利用该系统,通过用相应的DGC基因替换pleD,验证了来自苏云金芽孢杆菌和水稻黄单胞菌的两个推定的膜结合DGC具有功能。本报告首次报道了一种天然的三串联核糖开关,它被用作基于核糖开关的双荧光报告基因,用于在体内高效、方便地验证推定的DGC活性。