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Sci Rep. 2016 Feb 19;6:20871. doi: 10.1038/srep20871.

Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter.

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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.
Institute of Biochemistry, and NCHU Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.


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.

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