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.
Highlights
Pyrophosphate (TPP) riboswitches with similar structures that are adjacent to each other in B. anthracis12. 3) A SAM-II/SAM-V tandem arrangement riboswitch that senses S-adenosylmethionine (SAM) to allow for both transcriptional and translational control of downstream gene expression in “Candidatus Pelagibacter ubique”13. 4) Two independent riboswitches that are sequentially arranged to recognize separate ligands of SAM and coenzyme B12 in B. clausii[14]
In B. thuringiensis subsp. chinensis CT-4319,20, five c-di-GMP riboswitches are annotated in the Rfam database, with three of them arranged in tandem and possibly forming a triple-tandem riboswitch
While Bc1 and Bc2 are widely distributed in the B. cereus group[4], Bc3, Bc4, and Bc5 are connected in triple-tandem (Bc3-5 see Fig. 1 and supplementary Fig. S1) and only located in the 5′ -UTR of the cspABCDE mRNA in some B. cereus and B. thuringiensis (Supplementary Fig. S2)
Summary
Pyrophosphate (TPP) riboswitches with similar structures that are adjacent to each other in B. anthracis12. 3) A SAM-II/SAM-V tandem arrangement riboswitch that senses S-adenosylmethionine (SAM) to allow for both transcriptional and translational control of downstream gene expression in “Candidatus Pelagibacter ubique”13. 4) Two independent riboswitches that are sequentially arranged to recognize separate ligands of SAM and coenzyme B12 in B. clausii[14]. The chimera-type[38] or artificial ribozyme[39,40,41] incorporating c-di-GMP-I aptamer as the sensing domain have been applied as the in vivo c-di-GMP reporters These reporters are usually instrument-dependent, are inconvenient for routine DGC activity detections. We have constructed a novel reporter system comprising the naturally-occurring Bc3-5 fused between two fluorescence genes This novel reporter system was found to sensitively detect intracellular c-di-GMP level in vivo and reliably verify two membrane-bound putative DGCs under regular laboratory conditions without the need for extra expensive instruments
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