Expression And Purification Of SigmaK Protein And Identification Of Its Regulon In Bacillus Thuringiensis

Bacillus thuringiensis is a Gram-positive, spore-forming bacterium belonging to the Bacillus cereus group. It can produce not only spores, but also parasporal crystals with regular shape（ mainly encoded by cry and cyt genes）. Bt is the most widely used microbial pesticides, because it is highly effective aganinst target pests and environmentally friendly. In the process of spore formation, different Sigma factors play an important role in different space and time. Sigma K encoded by the sig K gene is an important factor during the late stage of sporultion. It involves in the important physiological processes, such as the formation of the exosporium, the maturation and release of the spores, the formation of crystals, mother cell lysis and so on. But little was known about those cellular process. Therefore this study mainly identify the regulon of Sigma K factor, which is the basis to understand the regulatory network of mother cell and clarify the molecular mechanism of spore formation in the late stage of sporultion.DNA microarray was performed to identify the genes or operons controlled by Sigma K factors using. RNA of sig K mutant and ger E mutant at T7 and T10（Tn represent n hours after the end of exponential phase） The data showed that 158 genes are down-regulated and 138 genes up-regulated at T7. while 878 genes are down-regulated and 1317 genes up-regulated at T10 in sig K mutant, compared with the global regulator ger E mutant. Using the consensus sequence of Sigma K factor binding sites, we found the 264 promoters of the down-regulated genes had the binding sites of the Sigma K, which direct the transcription of 10 operons and 242 genes. The functions of these genes include amino acid transport and metabolism, carbohydrate metabolism and transport, inorganic ion transport and metabolism, signal transduction, posttranslational modification, 216 other function genes and unknown genes.To verify the regulon of Sigma K factor, we expressed and purified Sigma K protein with His-tag. Previous study proved that the transcription of cry1 Ac gene is controlled by Sigma K factor. Electrophoretic mobility shift assays showed that Sigma K could bind to promoters of cry1 Ac. It suggests that purified Sigma K protein has a binding activity. Therefore the promoters of 6 genes（HD73₃496, HD73₃353, HD73₃410 and bxp B, exs B, bcl B）and the operon（HD73₂493, HD73₂494）were tested to bind with the purified Sigma K factor by EMSA. The results showed that Sigma K controlled the transcription of these genes. In additional HD73₃156 promoter fusions with lac Z were constructed to analyze the transcription activity of P3156。 The recombinant plasmids were introduced into B. thuringiensis strain HD73 and HD（?sig K）. The β-galactosidase assay showed that the activity of P3156 was abolished in sig K mutant. It further proved that P3156 is controlled by sK.