The Function Of Bacteria Enhancer Binding Proteins In Bacillus Thuringiensis

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium. Bt is generally distinguished from its relatives because of its ability to produce crystal inclusions. These crystals mainly consist of Cry proteins that are generally toxic for specific insects, thus Bt has been widely and successfully used as a biopesticide.Bacillus thuringiensis HD73strain is a representative strain of subspecies kurstaki strain. In our previous study, deletion of the sigL gene encoding the σ54factor decreased CrylAc protein production in Bt HD73strain,β-galactosidase assays of a fusion of the crylAc promoter and lacZ showed that the transcriptional activity of the crylAc promoter in the HD73wild-type strain did not differ from that in the sigL mutant. Therefore metabolic pathways regulated by the a54factor that do not affect crylAc gene transcription are important for CrylAc protein production.Sigma54-dependent transcription strictly requires activation by bacterial bacteria Enhancer Binding Proteins (bEBPs). In this study, we analyzed the functions of bEBPs in Bt HD73.We were able to identify eight proteins with Sigma54activator domains in the chromosome of B. thuringiensis HD73strain. They are generally consisting of three typical domains, the N-terminal regulatory domain, the central AAA+domain is responsible for sufficient to activate Sigma54-dependent transcription, and the C-terminal DNA binding domain contains a helix-turn-helix (HTH) motif. We analyzed the metabolic pathways of gene clusters containing these eight bEBPs. GabR is involved in y-Aminobutyric acid (GABA) pathway. SoxR is involved in sarcosine utilization. KamR is involved in lysine metabolic pathway. RocR is likely involved in arginine metabolic pathway. AcoR is likely involved in acetoin metabolic pathway. BkdR is likely involved in isoleucine and valine degradation pathway. LevR is likely involved in cellobiose and lichenase metabolic pathway. The metabolic pathway is regulated by PrdR is still unknown.We constructed the five bEBPs mutants by homologous recombination in Bt HD73. The growth curve showed that there is no significant difference between the mutants and HD73wild-type strain in LB medium and SSM medium, respectively. Motility analysis showed that the motility ability of sigL mutant strain, rocR, prdR, kamR, acoR, bkdR and levR mutants were decreased relative to that of HD73wild-type strain. However, the gabR and soxR mutants are no influence on motility. The sporulation efficiency showed that deletion of sigL gene sharply decreased the sporulation and rocR, kamR, and soxR mutants slightly decreased the sporulation relative to that of HD73wild-type strain. All the eight bEBPs mutants have no influence on Cry1Ac protein production.β-galactosidase assays showed that transcriptions of3213,0560,1070,4469and5613genes are activated by AcoR, RocR, PrdR, BkdR and LevR, respectively.The transcription unit of bkd gene cluster was identified by RT-PCR. The result showed that ptb-bkdB forms an operon. It regulated by Sigma54factor and activated by BkdR. The genetic complement of bkdR mutant recovered a part of motility, but the overexpression of bkdR is no influence on motility.