Expression, Purification And Function Of Bacteria Enhancer Binding Proteins In Bacillus Thuringiensis

Bacillus thuringiensis（Bt） is a member of Bacillus cereus group. Comparing with other Bacillus species, Bt can produce large crystalline parasporal inclusions during sporulation. Bt has been widely used as bioinsecticidal microbe for its high toxicity, safety and the other merits. The process of sporulation and crystal formation is related to metabolic patterns of different growth phase in Bt.In our previous study, we have been found that deletion of the sig L gene（encodes σ54 factor） decrease the efficiency of sporulation and reduce the production of crystal protein in Bt strain HD73. It indicated that the metabolic pathways, which are regulated by σ54, have a role in sporulation and crystal formation. The σ54-dependent transcriptions need to be activated by bacteria enhancer binding proteins（b EBPs）. We have been identified eight EBPs in Bt HD73, and purified Gab R and Sox R, which are involved in γ-aminobutyric acid（GABA） pathway and sarcosine utilization, respectively. In this study, the remaining six EBPs（Aco R, Bkd R, Kam R, Lev R, Prd R and Roc R） were cloned and expressed. We constructed the EBP genes with histidine（His） tags plasmids and successfully expressed in E. coli strain BL21. Bkd R and Lev R were purified by Ni-affinity chromatography.Herein, we analysed the transcriptional activity of the promoters of HD73₁024（encoding proline racemase）, HD73₂025（encoding branched chain amino acid aminotransferase）, HD73₄161（encoding proline peptidase two code）, HD73₄943（acetic acid coenzyme A ligase） and HD73₅327（encoding NADPH dependent dehydrogenase） in the eight EBP deletion mutants. b-galactosidase assays showed that transcription of HD73₄161 genes is activated by Gab R.Acetoin（3-hydroxy 2-butanone） is a major catabolic product of Bacillus grown aerobically in glucose media. The excretion of acetoin had its vital physiological meanings to these microbes mainly including avoiding acification, participating in the regulation of NAD/NADH ratio, and storaging carbon. Degradation products of acetoin are acetaldehyde and acetic acid, which is catalyzedby the acetoin dehydrogenase enzyme system in Bacillus subilis, Alcaligenes eutrophus and Klebsiella pneumoniae. In Bt, acetoin deradation is also catalyzed by the acetoin dehydrogenase enzyme system, which is encoded by aco gene clusters. The bioinformatic and RT-PCR analysis revealed that the aco gene cluster was composed of four genes（aco ABCL） and formed one transcriptional unit. The aco A promoter had conserved sequence-12/-24. The transcriptional activity of aco A promoter sharply decreased in sig L and aco R mutants, respectively. Deletion of aco R had no effect on growth and Cry protein production, but decreased the motility of cells and sporulation efficiency. It means that Aco R maybe control some genes controlled by σ54 to affect on the use of acetoin, motility and spore formation.