| Bacillus thuringiensis(Bt)is a kind of Gram-positive bacteria,which not only produces spores,but also produces toxin crystal proteins which have specific toxic effect on many kinds of insects.Most of crystal proteins production depends on spore development,so it is of great significance to study the molecular mechanism of spore development.In the whole process of spore formation,it is more important to determine the initial stage of sporulation.At present,it has been analyzed that SigmaH and its regulons played an important role in this process in Bacillus subtilis(Bs),but it is not clear whether the function of SigmaH in Bt is the same as that of Bs.This study mainly defines the regulons ofσ~H factor and studies the differential regulon compared with Bs,which lays a foundation for analyzing the regulatory network in the initial stage of sporulation in Bt.The reported SigmaH regulons in Bs include 54 transcriptional units(88 genes),which are mainly involved in cell metabolism and substance transport,cell signal transduction,cell division,sporulation,transcriptional regulation and so on.In this study,combined with transcriptome data and bioinformatics analysis,it was determined that the SigmaH regulons in Bt contained 278 transcriptional units(374genes).Comparative analysis showed that there was no significant difference in the functional classification of SigmaH regulons between Bt and Bs,but there were significant differences in regulons under each classification,only the regulons of sporulation class were more conservative.Transcriptional activity analysis and gel retardation test have been used to verify that sporulation genes such as spo0A,spoVG,spoVS1 and spoVS2 in Bt are also controlled by SigmaH.Based on the analysis of the differences between 12 regulons of sporulation class in Bt and those in Bs,it was found that spoVS1 and spoVS2 were different from those in Bs,and both of them were homologous to spoVS gene in Bs.In order to determine whether there were differences in sporulation,the functions of these two genes were explored in Bt.Transcriptional activity analysis showed that the expression patterns of spoVS1 gene and spoVS2gene were similar before forespore engulfment,but the transcriptional activity of spoVS2 gene was lower than that of spoVS1 gene.Further clarifying their biological functions,it was found that the deletion of spoVS1 gene would delay the formation of polar septum,produce bipolar septum,hinder the maturation and release of spores,and significantly decrease the sporulation frequency and Cry1Ac protein production.The deletion of spoVS2 gene did not affect the formation of polar septum,delayed the release of spores,did not affect the sporulation frequency,but decreased the Cry1Ac protein production.By comparing the functions of spoVS1 and spoVS2 genes in Bt with spoVS genes in Bs,it was found that the function of spoVS1 gene was similar to that of spoVS gene in Bacillus subtilis,while the function of spoVS2 gene was different from that of the two genes.The functions of spoVS1 gene and spoVS2 gene in the process of polar septum formation and spore maturation are partially redundant,but there are differences in other aspects.According to the phenotypic analysis of spoVS1 gene and spoVS2gene mutants,it was found that the deletion of spoVS1 gene and spoVS2 gene could inhibit the transcriptional level of sigE gene in different degrees.NCBI BlastP results show that SpoVS1 and SpoVS2 are widely distributed in the Bc group and are highly conservative.In this study,theσ~H regulons of Bt were identified,and the function ofσ~H regulon which was different from that of Bs was studied.It is helpful to clarify the gene function in the specific regulatory module of Bt and lay a foundation for the analysis of regulatory network in the initial stage of sporulation in Bt... |
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