| Bacillus thuringiensis(Bt)is one of the most widely used microbial insecticides.However,Bt formulations still have shortcomings such as relative short field duration due to ultraviolet(UV)radiation.Bacterial biofilms(BBFs)can enhance the ability of bacteria to resist UV radiation and other uncomfortable environments.The analysis of the regulation of important genes on BBFs associated phenotypes can lay the foundation for improving the UV resistance of Bt and constructing highly UV-resistant Bt engineering bacteria.In the previous work of our research group,we obtained important regulatory proteins and genes in the process of Bt XL6 biofilm formation and disperal through comparative proteogenomics.In this paper,two candidate key genes,BTXL6＿06815and BTXL6＿14170,were selected as the research objects,and their regulation of Bt XL6 biofilm associated phenotypes was studied.The main findings are as follows:(1)The changes of transcription level and protein expression level of BTXL6＿06815 and BTXL6＿14170 genes during biofilm formation of Bt XL6 were clarified.Compared to planktonic cells(18 h),the BTXL6＿06815 gene was significantly up-regulated in the biofilm adhesion phase(20 h),the mature phase(34 h)and the dispersal phase(56 h)at the transcription level;the BTXL6＿14170 gene was significantly up-regulated in the biofilm adhesion phase(20 h)and the mature stage(34 h)at the transcription level.The results of proteomics are consistent.These indicated that the BTXL6＿06815 and BTXL6＿14170 genes may regulate the biofilm formation of Bt XL6.(2)Bioinformatic analysis indicated that BTXL6＿06815 may be S-ribosylhomocysteinase(LuxS),and BTXL6＿14170 was likely to encode a histidine kinaseBLAST analysis of deduced amino acid sequences of BTXL6＿06815 and BTXL6＿14170 showed that 99% identities with Bacillus S-ribosylhomocysteinase(LuxS)and histidine kinase,respectively.These two genes are free of transmembrane domains and signal peptides.Predictive analysis found that the BTXL6＿06815 gene may be regulated by the transcription factors Cod Y,Sig E and Sig H,while the BTXL6＿14170 gene may be controlled by the transcription factors Sig F and Deg U.(3)BTXL6＿06815 and BTXL6＿14170 genes positively regulated the biofilm formation of Bt XL6The gene knockout mutants of BTXL6＿06815 and BTXL6＿14170genes and their complementary strains were constructed.Using confocal laser scanning microscope,the thickness of Bt XL6 BBF is about 43.2280± 4.3104 μm,which was significantly thicker than those of BBFs of Bt XL6△06815ΩKm(35.1613± 5.1976 μm)(p<0.01)and Bt XL6△14170ΩKm(35.8380±3.3082 μm)(p<0.01).This indicated that BTXL6＿06815 and BTXL6＿14170 positively regulated the biofilm formation of Bt XL6.(4)BTXL6＿06815 and BTXL6＿14170 genes positively regulated the UV-B resistance of Bt XL6In the state of planktonic cells,knockout of the BTXL6＿06815 gene resulted in a significant decrease in the UV-B resistance of cells in the vegetative and lysis phases,and did not affect the UV-B resistance of cells in the sporangial phase.In the BBF state,knockout of the BTXL6＿06815 gene resulted in a significant decrease in the ability of cells to resist UV-B in all three phases.Under the condition of planktonic cells,knockout of the BTXL6＿14170 gene resulted in a significant decrease in the ability of cells to resist UV-B in all three phases.In the BBF state,knockout of the BTXL6＿14170 gene resulted in a significant decrease in the resistance of cells to UV-B in the vegetative and lysis phases.(5)BTXL6＿06815 is a key factor in the resistance of Bt XL6 to polymyxin B,while BTXL6＿14170 gene negatively regulated the maltotriose production in Bt XL6After the BTXL6＿06815 gene was knockout,the resistance of Bt XL6 to polymyxin B changed from positive to negative.Polymyxin B is mostly used in clinical treatment of severe infections and has antibacterial activities against most strains.When bacteria form BBFs,drug resistance may increase significantly.When the BTXL6＿06815 gene was knockout,the amount of BBF formed from the Bt strain was reduced,and the drug resistance was weakened.Therefore,the BTXL6＿06815 gene knockout mutant was not resistant to polymyxin B.On the other hand,knockout of the BTXL6＿14170 gene changed the strain’s biochemical identification result of maltotriose from weakly negative to weakly positive without known mechanisms.(6)The BTXL6＿06815 and BTXL6＿14170 genes did not affect the growth,sedimentation and cell motility of Bt XL6The knockout of BTXL6＿06815 and BTXL6＿14170 genes did not affect the growth trend of the strains.After standing for 24 h,all Bt strains settled to the bottom of the test tubes,indicating that knockout of the BTXL6＿06815 and BTXL6＿14170 genes did not affect the sedimentation ability of Bt XL6.After cultured in LB solid media with0.3%,0.5% and 0.7% agar for 12 h and 24 h,there was no significant difference in colony radius between Bt strains,indicating that these two genes did not affect cell motility.(7)Heterologous expression of BTXL6＿06815 and BTXL6＿14170 genes in E.coli laid the foundation for the determination of protein activitiesUsing IPTG induction,20 k Da BTXL6＿06815 protein and 43 k Da BTXL6＿14170 protein were expressed in E.coli,which provided materials for activity identification.In summary,this thesis clarified the changes in the transcription level and protein expression level of BTXL6＿06815 and BTXL6＿14170genes during the biofilm formation of Bt XL6,speculated that BTXL6＿06815 may be S-ribosylhomocysteinase(LuxS)and BTXL6＿14170 may be a histidine kinase,drew conclusions that these two genes positively regulated the biofilm formation and UV-B resistance of Bt XL6 based on gene knockout and determination of biofilm associated phenotypes.Further researches indicated that BTXL6＿06815was a key factor for the resistance of Bt XL6 to polymyxin B,and BTXL6＿14170 gene negatively regulated the maltotriose production in Bt XL6.These results laid a solid theoretical foundation for the in-depth study of regulatory network of biofilm formation and the construction of anti-UV Bt engineering strains. |
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