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

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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Section B: Additional result figures Figure S1. Sequence of triple-tandem c-di-GMP-I encoding region and its downstream operon. (a) Sequence of the triple-tandem c-di-GMP-I Bc3, Bc4 and Bc5 RNA were shown in red, lime and blue colors, respectively. Linkers between the adjacent riboswitches were shown in black color. Aptamer sequences were underlined by solid lines, and the region between two gray triangles was an omission in GenBank record (GenBank accession: NC_017208.1) due to a splicing error.

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Section C: Primers information and plasmids construction procedure.  (Table 1), and transformants were verified by sequencing. Plasmids were constructed as follows:

Construction of plasmids for β-galactosidase assay in E. coli Trans5α with various c-di-GMP riboswitch encoding regions in promotors
Gene lacZ was cut (fragments between Hind III and Kpn I) from our previously engineered plasmid pHT1K-lacZ(UTR) 3

Construction of plasmids for PleD expression in E. coli
A previous reported DGC PleD 5 was used for the synthesis of intracellular c-di-GMP. Amplification of its encoding gene pleD (primer pair: pleD_F and pleD_R) was carried out using a gift PleD expression plasmid 6 as a template, and then fused (between Xba I and Sal I) into pET-28b(+) (Novagen, catalog S11 number: 69865-3) to form pET-28b(+)-pleD (Supplementary Figure S7). Subsequently, a fragment carrying Ptac promoter was amplified (primer pair: Ptac_F and Ptac_R) from commercial plasmid pGEX-6P-1 (GE Healthcare Inc., code number: 28-9546-48) to replace the T7 promoter (between Bgl II and Xba I) of pET-28b(+)-pleD, thus the PleD constitutive expression plasmid pET-Ptac-pleD (Supplementary Figure S7) was generated. Plasmid pET-28b(+) was used as a negative control.

Construction of riboswitch-based dual-fluorescence reporter plasmids used in E. coli BL21(DE3)
PleD inducible expression plasmid pET-28b(+)-pleD (Supplementary Figure S7) was constructed as above description and used for the regulation of intracellular c-di-GMP level, while pET-28b(+) was used as a negative control. Riboswitch-based dual-fluorescence reporter plasmids were constructed by fusing riboswitch encoding regions between amcyan (a cyan fluorescence protein gene from Anemonia majano) and turborfp (a turbo red fluorescence protein gene from Entacmaea quadricolor).
For construction of pRP0122-Pbe-amcyan_Bc3-5_turborfp, a backbone plasmid pRP0122 (Supplementary Figure S8) was firstly generated. In fact, pRP0122 was the truncated derivative of pRP1028, in which turborfp gene and Pbe were deleted. To obtain pRP0122, pRP1028 was cleaved with Pvu II and EcoR I to generate a linearized pRP1028, where the DNA region containing turborfp gene, spectinomycin resistance gene and Pbe was removed. Then, the spectinomycin resistance gene was amplified from pRP1028 using primers speR_F and speR_R (Supplementary Table S6) and ligated to the linearized pRP1028 at Pvu II and EcoR I sites, finally resulting in the formation of pRP0122. Subsequently, amplification of Pbe (primers: Pbe_F and Pbe_R), amcyan (primers: amcyan_F and amcyan_R), Bc3-5 (primers: Bc3_F and Bc5_R) and turborfp (primers: turborfp_F and turborfp_R) were carried out using pRP1028, pSS4332, CT-43 genome DNA and pRP1028 plasmid as their corresponding templates, respectively. Plasmid pSS4332 was a gift from Scott Stibitz (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA). Next, they were assembled with blunt ends through overlap extension PCR, then digested with Kpn I and ligated with a linearized pRP0122 which was cleaved with Sma I and Kpn I. The constructed plasmids were transformed to BL21(DE3) for Congo red assays and expressed proteins of putative DGCs and their variants were subsequently purified (Table 1). They are co-transformed to BL21(DE3) with pRP0122-Pbe-amcyan_Bc3-5_turborfp to construct the strains for putative DGCs verification (Table 1).  kanamycin resistance gene; pBR322: pBR322 replicon; lacI: lactose repressor gene.