Dataset Information

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.

SUBMITTER: Zhou H

PROVIDER: S-EPMC4759541 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Zhou Hang H Zheng Cao C Su Jianmei J Chen Bo B Fu Yang Y Xie Yuqun Y Tang Qing Q Chou Shan-Ho SH He Jin J

Scientific reports 20160219

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 show ...[more]

PMID: 26892868

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Project description:High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein.In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.

Dataset Information

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

Publications

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

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