Function Of Two C-di-GMP Riboswitches In Bacillus Thuringiensis BMB171

Cyclic diguanosine monophosphate(c-di-GMP)is one kind of nucleotide-based second messenger in bacteria,synthased by DGC(diguanylate cyclase)with GGDEF domain and degraded by PDE(phosphodiesterase)with EAL or HD-GYP domain.Binding to downstream receptors or targets including riboswitch as the most specific one,c-di-GMP can regulate a variety of biological processes,such as virulence,motility and biofilm formation.Riboswitches are located primarily within the 5'-untranslated region(5'-UTR)of the main coding region of a particular mRNA and composed of two domains:a natural aptamer and an expression platform and enormously widespread among bacteria,fungi and plants.Small molecule such as glycin and guanylic acid binds to the aptamer,inducing the conformational change in expression platform which regulates the downstream gene expression.The c-di-GMP riboswitches are discovered in recent years and widespread in bacteria.At present more attention are paid to their structures but little to the downstream gene function.Sequence analysis shows that there are two known c-di-GMP riboswitches and many c-di-GMP signaling genes in Bacillus thuringiensis BMB171 genome.Both the two c-di-GMP riboswitches utilize termination-antitermination to regulate downstream genes'transcription according to the analysis of their secondary structures.Further study on the regulatory mode and downstream gene function of the c-di-GMP riboswitches will throw light on the role of the c-di-GMP riboswitches.We constructed transcription-fused vectors of the 5' regions of both c-di-GMP riboswitches via sequence truncation,riboswitch-deletion and transcriptional fusion.And all the vetors were introduced into B.thuringiensis BMB171 by electrotransformation.Then the ?-galactosidase activity assay was conducted.Through the comparison of the transcription activity of inserted segements,the minimal reglation region and the function of c-di-GMP riboswitches can be deduced.C0363 and C0936-knockout mutants BMB171?C0363 and BMB171?C0936 were obtained by I-SceI mediated gene-knockout.And the biofilm formation,strain morphology,sporulation and parasporal crystal formation were analyzed.Moreover,the chemotaxis of BMB171 ?C0363 and the adhesion between collagen adhesion protein encoded by C0936 and collagen were studied.The minimal transcription regulatory region of C0363 is located in 561 bp before its start codon while that of C0936 is still uncertain because of the low transcription activity of its 5' region.The two genes' promoters harbor high transcriptional activity which decrease sharply when connected with the riboswitches.The c-di-GMP riboswitches regulate C0363 negatively and C0936 positvely showed by secondary-structural analysis of c-di-GMP riboswitches.BMB171 ?C0363 and BMB171?C0936 grow normally,form spore and parasporal crystal while their ability of biofilm formation decrease.Preliminary results show that the chemotaxis toward proline of C0363-knockout mutant is impaired;collagen adhesion protein encoded by C0936 can bind collagen.We prove that the c-di-GMP can regulate downtsream gene expression via riboswitches.Both C0363 and C0936 can affect biofilm formaion.Biofilm formation is one of the key indicators for the bacterial virulence,so this study can offer new clues to improve the insecticidal activity of B.thuringiensis.