Study On Regulation Of Transcriptional Regulator CodY In Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that can form spores and parasporal crystals during the stationary phase. It plays an important role in the prevention and control of insects including lepidoptera, diptera, coleoptera, hymenoptera and nematodes. CodY is a highly conserved protein in low G+C Gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. On the basis of identification of the differential proteins of BMB171codY-cry1Ac and BMB171cry1Ac and CodY target genes in vitro experimental technique, we carried on the transcription level validation by fluorescent quantitative PCR and performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulatory genes of CodY. The detailed and in-depth analyses of the regulating of CodY establish the foundation for highly efficient and stable expression of parasporal crystals in B. thuringiensis and also offer a potential biocatalyst for functions of CodY in other pathogenic bacteria.1. According to the analysis of the growth, morphological and physiological changes of codY mutant B. turingiensis strain BMB171codY-cry1Ac and comparative proteomic analysis, we carried on the transcription level validation by fluorescent quantitative PCR. The genes were cotN which was involved in sporulation, phap, crt, gabT, acsA involved in biosynthesis of poly-β-hydroxybutyrate, protease related genes calY, hslV, nprB. The results were consistent with2-DE data.2. On the basis of two-dimensional electrophoresis and MALDI-TOF-MS/MS analysis, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments with some sequences such as promoter region of frr operon, promoter region of codY operon, internal sequence of hslU, upstream sequence of comER, promoter region of phaP, promoter region of ilv-leu operon and confirmed the direct regulation of genes by CodY. The results showed that the proteins regulated by CodY are involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence and BCAAs metabolism. We firstly reported that CodY could directly regulate ribosomal recycling factor FRR and may influence production of parasporal crystals in B. turingiensis.3. According to the results of CodY target genes in vitro experiment, we analyzed the binding motif of CodY using bioinformatics technology and researched the important functional protein. The results showed that proteins involved in metabolism, oligopeptide permease OppA, recombinase RecA, two-component signal transduction system LytSR and VanSR and some aminopeptidases were regulated by CodY protein.Combining with analysis of CodY regulated genes both at the protein level and at the genetic level; we found that CodY regulates many genes mainly involved in microbial metabolism, such as amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, energy metabolism. Furthermore, the proteins involved in growth, sporulation, protease, transport, two-component signal transduction, genetic competence were regulated by CodY.