Transcriptional Factor CpcR Regulates The Expression Of Cry Genes In Crystal-Producing Cell

A challenge in bacterial developmental biology is to understand the mechanisms underlying cell fate decisions.Cell differentiation within isogenic population allows specialization of subpopulations and an efficient division of labor which contributes to the survival of bacteria under unfavorable conditions.Bacillus thuringensis is a spore-forming bacterium that produces insecticidal crystal proteins in the sporulating cells.B.thuringiensis LM1212 strain displays a unique phenotype which can mainly differentiate into two distinct subpopulations,one of which forms spores without producing crystals while another produces crystals without forming spores.Previous experimental results showed the crystal producing cell regulator?CpcR?may affect crystal inclusion production in the differentiated cells.In this thesis,the regulation of cry gene in the differentiated cells by CpcR was studied,and the main contents are as follows:The transcriptional start site of cpcR gene was determined by 5?-RACE methods.The cpcR gene was regulated by two promoters:a proximal CpcR-regulated promoter?P1 region?generating a positive loop of regulation particularly effective during stationary phase;a distal promoter?P2 region?with a weak and constitutive transcriptional activity.There was a 30-bp conserved motif in the promoter region of LM1212four cry genes,as well as in the promoter region of cpcR gene.To determine the importance of the conserved motif,nucleotide substitutions?ACT vs TGA or GT vs CA?were introduced in the promoter of cpcR and cry35-like gene.The results indicated that in the HD73 strain,the expression of lacZ reporter gene and fluorescent gene directed by the mutated promoters were almost abolished in the presence of CpcR.Electrophoretic mobility shift assays suggested that CpcR directly bind to target DNA with a low affinity,but nucleotide substitutions did not affect the binding ability of CpcR.CpcR belongs to response regulator of OmpR family.According to the gene annotation results,the two-component histidine kinase encoded by orf32 gene may be involved in the phosphorylation process of CpcR.However,our results showed that CpcR was able to activate the expression of P_cry35?lacZ similarly no matter orf32 was presence or not.In addition,BLASTP analysis revealed that the genome of HD73 strain harbored a gene encoding an orthologous gene of Orf32 with high identity,HD73?orf2857.When we deleted the gene HD73?orf2857 in HD73 strain,the expression kinetics of both P_cry35?lacZ and P_cpcR?lacZ were similar in the wild-type and mutant strains in the presence of CpcR.These results showed CpcR was active in the HD73 strain independently of the kinase Orf32 or HD73?2857.In addition,Spo0A,SinI and SinR had no effect on CpcR transcription,but CodY presented a negative effect on CpcR transcription in the exponential phase.CpcR was able to use P_cry35 to direct the production of a crystal inclusion encoded by insecticidal gene cry1Ab in non-sporulating cells of HD73 strain.Moreover,cpcR expression induced a strong reduction in sporulation.In this thesis,the relationship between CpcR and sporulation-related genes was preliminarily explored.The yfp/mCherry dual-labling analysis indicated that most cells began to express cpcR gene from the period T0 and the expression gradually increased from period T2 to T4.When CpcR existed,the expression pattern of mCherry gene induced by P_spo0A was similar to that of cpcR gene,but a few cells only express mCherry gene;P_spoIIE-directed mCherry gene expressed from T2 period,and only a small number of cells expressed mCherry gene or yfp gene,but most cells expressed both the two fluorescent genes;The expression of mCherry gene directed by P_spoIID started from period T4,and a small number of cells only expressed yfp gene,while most cells expressed both the two fluorescent genes.However,P_spoIID-directed mCherry gene began to express in T3 period in the absence of cpcR gene,which indicated that the expression of cpcR gene delayed the transcription of P_spoIID.