title: Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter.
abstract: 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Hang
authors.initials: H
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Cao
authors.initials: C
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Jianmei
authors.initials: J
authors.lastname: Su
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), College of Bioengineering, Hubei University of Technology, Wuhan, Hubei 430068, People's Republic of China.
authors.forename: Yuqun
authors.initials: Y
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Institute of Biochemistry, and NCHU Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Shan-Ho
authors.initials: SH
authors.lastname: Chou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
authors.forename: Jin
authors.initials: J
authors.lastname: He
mesh_terms: D001483:Base Sequence; D019295:Computational Biology / Q000379:methods; D006152:Cyclic GMP / Q000031:analogs & derivatives*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D058928:Riboswitch
chemical_list: D012333:RNA, Messenger; D058928:Riboswitch; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP