Functional Analysis Of Orf28-32 Fragment Harboring In The Large Plasmid Of Bacillus Thuringiensis LM1212 Strain In HD73 Strain

Bacillus thuringiensis is a gram-positive bacterium,belonging to Bacillus cereus.For traditional Bt strains,the most prominent character is that the insecticidal crystal proteins(ICPs)were produced in the same mother cell during the process of forming spores,which indicated that more energy would be consumed.Interestingly,a novel Bt strain LM1212 was found to produce ICPs in the cells without spores,thus,cells could produce more ICPs by using the abundant energy and matter.In this paper,we focused on the study about the transcription factors involved in the process of crystal cell differentiation and tried to identify the functions of these transcription factors in a gram-positive type strain,HD73,which may lay a solid foundation for the application of other Bt strains.Our previous study showed that the orf28-29 fragment located in the plasmid pLM248 in LM1212 strain,which could improve the proportion of crystal cells.In our work,we studied the biological function of orf28-29 fragment in HD73 strain and tried to find the interactive genes.After having been integrated into HD73 genome,our results showed that the number of spores increased and the crystal cells decreased in HD73,however,the change was not significant.So,we speculated that there may some other genes were also involved in this process.Bioinformatics analysis revealed that orf28 encoded a crystal producing cell regulator(CpcR),and its downstream gene orf32 encoded the sporulation kinase E.Structural analysis revealed that orf28 and orf32 both had the typical structures of the two-component signaling system,so we inferred that orf28 and orf32 formed a two-component signaling system together to regulate the process of crystal cell differentiation.The p35'gfp fragment was integrated into amyE locus of HD73 strain to construct the mutant strain HD(p35'gfp).The orf28-29 fragment and orf28-32 fragment were integrated into pHT304,a high copy number vector,to form a new recombinant plasmid,which was transferred into the mutant strain to study its function.Laser confocal microscope observation illustrated that the amount of spores decreased and crystal cells increased in the mutant,which meant that the influence of orf28-29 fragment was related to its copy number.The sporulation efficiency analysis showed that the total number of cells decreased significantly after pHT304 recombined with orf28-29 fragment or orf28-32 fragment was introduced into HD73 strain,which suggested that orf28-29 fragment and orf28-32 fragment could decrease the number of living cells.Laser confocal microscope observation experiment also showed that there were three different cell subpopulations in HD73 strain containing the pHT304 plasmid connected with orf28-29 fragment or orf28-32 fragment respectively : the cells producing crystals without forming an asymmetric membrane,the cells differentiating into crystal producers after forming an asymmetric membrane and the cell forming spores with engulfment after forming an asymmetric membrane.It also showed that orf28-29 fragment and orf28-32 fragment dramatically influenced the process of spore engulfment.In this study,the functions of orf28-29 fragment and orf28-32 fragment were verified and the novel transcriptional regulation mechanism of crystal cell differentiation was studied in HD73 strain.It was promising to develop a novel non-spore system of cry gene expression for the molecular design of new generation Bt-insecticide.