Research On The Function Of Dlt Operon In The Interaction Of Bacillus Thuringiensis And Insect

Most gram-positive pathogenic bacterial,such as Bacillus anthracis,Listeria monocytogenes,Streptococcus pneumoniae and Staphylococcus aureus,harbor dlt operon responsible for modification of the teichoic acids in cell wall by D-alanylation.This modification confers net positive charge in cell surface,and results in pathogenic bacterial resistance towards the host cationic antimicrobial peptides(AMPs)by escaping the host immune response.This is an effective protective mechnism for pathogenic bacterial,and is crucial for its survival in natural environment.Bacillus thuringiensis(Bt)is a gram-positive pathogenic bacterium of insects and nematodes.We analyzed its genome and found that there is also a dlt operon.As an insecticidal microorganism,Bt kills insect mainly dependent on insecticidal crystal protein.In the interaction of Bt and insect,they evolved diverse protection mechanism,respectively.The dlt operon is likely an effective way for Bt to escape the insect immune response.In the current study,the effect of dlt operon on the growth and reproduction of Bt was analyzed by knocking out it,and its biological function in the interaction with insect host was elucidated.In this research,the dltA gene in acrystalliferous mutant BMB171 was knocked out by homologous recombination technique,and the loss-of-function mutant of dlt was successfully constructed.The result of surface charge difference analysis showed that the loss-of-function of dlt could cause a significant increase in the net negative charge of the cell surface,and verified that it also functions in regulating the surface charge of the cell in Bt.Morphological observation showed that the loss-of-function of dlt could lead to a significance reduction in cell biomass and retard sporulation,but no significant change in bacterial morphology was observed.This result indicated that the dlt operon could affect the growth and reproduction of cells and indirectly regulate the generation of the parasporal crystal.Further study indicated that the sensitivity of the mutant to AMPs is significantly enhanced,indicating that the dlt operon could enhance the resistance of Bt to AMPs and,which is conducive to escape the insect immune response.The resistance analysis showed that that the dlt operon did not change the tolerance of the Bt to temperature,but the loss-off-function could increase tolerance to acid,while attenuated tolerance to alkali.This suggests that the dlt operon can prolong its survival time in the intestine of the insect by changing the pH tolerance,thus help to enhance the insecticidal virulence.The cell experiments show that the dlt operon could escape the insect immune defense by attenuating the adhesion of insect cells to Bt.In summary,the results of this research show that dlt operon plays an important role in the interaction between Bt and insect,which is an effective protective mechanism for Bt to resist insect host immune response.It will help us to understand the importance of dlt operon for the survival of Bt in nature habitat on the perspective of ecology,and provided a new strategy for improving its ability to infect insect hosts and to cope with insect potential resistance.