The Mechanism Of Transcription Factor CpcR Regulates The Initiation Of Sporulation

Bacillus thuringiensis is a gram-positive bacterium capable of differentiating into a spore.During sporulation process,this bacterium produces insecticidal toxins in the form of a crystal inclusion,usually in the sporulating cell.We previously reported that Bacillus thuringiensis LM1212 strain can differentiate into two distinct subpopulations of spore formers and crystal producers.This division of labour phenotype provides it with an advantage in competition with a typical Bacillus thuringiensis strain HD73.In the further study,we found that the transcription factor CpcR was characterized as the major regulator responsible for the cell differentiation of Bacillus thuringiensis strain LM1212.However,the CpcR regulatory network is unclear.In this research,we examined how CpcR affects sporulation network.This study emphasizes the complexity in the regulation of sporulation and illustrate the diversity in the strategies employed by bacteria to ensure their survival,and provides an important theoretical basis for the construction of a new generation of spore-free engineering strains.The main research contents are as follows:1.The effect of CpcR on sporulationMicroscope observation and spore count of the strains in the presence of cpcR indicated that CpcR inhibited sporulation.Microscope observation of the strain stained with FM 4-64 and MTG indicated that the process of sporulation was inhibited before the formation of the polar septum in the presence of cpcR in LM1212 strain,and was blocked in engulfment in the presence of cpcR in the HD73 strain.The transcriptional analysis of spo0A promoters based on lac Z reporter indicated the transcription of spo0A was increased in presence of cpcR.The transcriptional analysis of spo IIE promoters suggested that the activity of Spo0A was reduced in the presence of cpcR in LM1212 strain,and was enhanced in the presence of cpcR in the HD73 strain.The overexpression of spo IIE gene showed that its overexpression could reduce the production of spore.2.The Screening of CpcR-regulated geneA spo0E family gene(spo0E1)was identified by comparative analysis of genes whose promote contained CpcR-box in LM1212 strain and HD73 strain.The transcription start sites of spo0E1 gene were determined by 5’-RACE method.The transcriptional analysis of spo0E1 gene promoter based on lac Z reporter showed that CpcR positively regulated the transcription of spo0E1 gene.3.CpcR modulates the production of spores by Spo0E1Overexpression of spo0E1 gene under the control of xylose-inducible promoter showed that Spo0E1 was indeed a negative regulator of sporulation.Spore count of the strains in the presence of both cpcR and spo0E1 gene suggested that CpcR affected the product of spores by modulating the spo0E1 expression.Spore efficiency of the strains in which expression level of spo0E1 is different showed that variations in the level of spo0E1 expression modulate the production of spores.4.Single-cell analysis of cpcR expressionThe mcherry/yfp dual-labeling analysis of cell differentiation in LM1212 strian indicated that both cpcR and spo0E1 were specifically expressed in crystal producers,and cpcR and spo IIE were expressed in different subpopulation.5.The exploration of regulatory factors related to activity of CpcRThe transcriptional analysis of cry35-like gene promoter in the sig H gene mutant strain or spo0A gene mutant strain indicated the activity of CpcR requiredσ~H and was repressed by Spo0A.