A Study On Two-Component Singal Transduction Systems Of Bacillus Thuringiensis Strains

Bacillus thuringiensis (Bt) is widely used as biological pesticides. It is widespreadly present in various environments and can synthesize insecticidal crystal proteins to form parasporal crystals during sporulation period. However, its rapid adaptation to the changing environments and the mechanism of parasporal crystals formation remain unclear. In this study, we deeply analyzed all the two-component signal transduction system (TCS) of three Bt strains YBT-1520, CT-43 and BMB171 by bioinformatics method. The typical TCS YycGF was selected to further study its operon structure and function. The aim of this study was to provide a novel direction for the study the growth, metabolism, expression, and regulation of toxic genes as well as the formation mechanism of parasporal crystals in Bt.We comprehensively analyzed the distributions, structures and putative biological functions of TCS from the genomes of the three Bt strains which were sequenced by our own laboratory. And more importantly, we preliminarily constructed the TCS regulatory networks.Three protein expression strains BL21(DE3)/pET28b-yycG, BL21(DE3)/pGEX-6P-yycH and BL21(DE3)/pGEX-6P-yycI were constructed. The corresponding proteins YycG, YycH and YycI were expressed and purified by Ni-NTA and GST resins, respectively.To reveal the interactions between YycG and YycH and YycG and Yycl, pull-down and bacterial two-hybrid experiments were performed. The results indicated that the interactions between both of them were confirmed, furthermore, the later was stronger than the former.The overexpression recombinant strain of response regulator gene yycF was constructed. The yycF was cloned into shuttle vector pHT1K-Plip-TS to gain the overexpression vector pHT1K-yycF. Then the recombinant strain named BMB/F was obtained by transforming the plasmid pHT1K-yycF into BMB171. RT-PCR results indicated that the transcription level of yycF gene was higher in the recombinant strain than that of original strain. SDS-PAGE results showed that the expression level of the total protein were obviously different between recombinant strain and original strain.