Functional characterization of cpcR-c1 and cpcR-t, homologous genes of transcription factor gene cpcR

Abstract:

Compared with typical strains of Bacillus thuringiensis (Bt), LM1212 strain can differentiate into spore-formers and crystal-producers. In LM1212, the crystal-producing cell regulator (CpcR) not only participates in cell differentiation but also activates the promoter of crystal protein genecry35-like (P₃₅). [Objective] We aimed to screen out the homologous genes of cpcR and verify their biological functions. [Methods] We cloned two cpcR homologous genes, cpcR-c1 from Bacillus cereus and cpcR-t from B.toyonensis. Then, we inserted cpcR and its homologous genes into pHT304-P₃₅-gfp and pHT304-P₃₅-lacZ vectors, respectively. The recombinant plasmids were transferred into Bt HD73⁻ strain without cpcR and the crystal protein gene. We then observed the cell phenotypes of recombinant strains HD⁻(cpcR-c1-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-gfp) by using a laser confocal microscope and quantified the sporulation efficiency. The β-galactosidase activities of HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ) strains were determined. [Results] Compared with the control strain, strain HD⁻(cpcR-c1-P₃₅-gfp) and HD⁻(cpcR-t-P₃₅-gfp) showed the number of spores decreasing by 80.79% and 90.14% and the percentage of crystal-producers increasing by 7 and 9 times, respectively. Gene gfp was expressed in these two strains. The β-galactosidase activity assay demonstrated that promoter P₃₅ had high transcriptional activity in strain HD⁻(cpcR-c1-P₃₅-lacZ) and HD⁻(cpcR-t-P₃₅-lacZ). [Conclusion] The homologous genes of cpcR, cpcR-c1 and cpcR-t can regulate cell differentiation and activate the transcription of P₃₅, and cpcR-c1 had better performance in activating P₃₅ transcription than cpcR.