Effect of Sigma H on sporulation in Bacillus thuringiensis

Abstract:

[Objective] To test the regulation of spo0A transcription by the regulatory protein Sigma H (σ^H) from Bacillus thuringiensis (Bt) HD73;Expressing and purifying Sigma H in Escherichia coli to verify its direct binding to the promoter of spo0A;To detect the effect of deletion of sigH on production of spores and crystal proteins in B.thuringiensis HD73.[Methods] The transcription level of spo0A was determined by measuring the β-galactosidase activities directed by the promoter of spo0A in Bt HD73 and sigH deletion mutant.The open reading frame of the sigH was amplified by PCR from Bt HD73 genome,and then ligated into the vector pET21b to generate pETsigH.The pETsigH was transformed into BL21(DE3) to obtain the recombinant strain BL21(pETsigH).The Sigma H protein was extracted and purified by Ni Sepharose 6 Fast Flow column purification and anion exchange purification.The electrophoretic mobility shift assay (EMSA) was carried out to verify the direct binding of Sigma H and the promoter of spo0A.The phenotypic characteristics of the sigH deletion mutant strain (HDΔsigH) were analyzed by microscopic observation and sporulation efficiency determination. [Results] The deletion of sigH decreased the transcription level of spo0A.The 28 kDa-Sigma H-His was expressed and purified from E.coli strain.EMSA results showed that the Sigma H-His could bind to the promoter of spo0A.Microscopic observation and sporulation efficiency determination demonstrated that the HDΔsigH failed to produce spores and crystal proteins.[Conclusion] The Sigma H-His protein directly regulates the expression of spo0A by binding to the promoter of spo0A.Deletion of the sigH blocks the spores and crystal proteins production in Bt strains.