Omics Analysis Provides Insight Into The Key Regulatory Proteins And Temporal Expressed Genes Involved In Bacillus Thuringiensis Biofilm Formation And Dispersion

Bacillus thuringiensis(Bt)is one of the most widely used and most successful microbial pesticides,but it is vulnerable to external environmental factors(such as ultraviolet,high temperature,drought,etc.),which cause relative short period of field duration and limit its further development.Bacterial biofilm(BBF)is a kind of protective growth method for bacteria to adapt to the environment,which can improve the ultraviolet resistance of bacteria,and enhance the ability of bacteria to adapt to environmental stress.This feature provides a new solution to break the bottleneck,the short duration of bacterial pesticide field.Based on omic techniques,key regulatory proteins and temporal expressed genes involved in Bacillus thuringiensis(Bt)biofilm formation and dispersion were studied,which was helpful for the development of Bt product with longer field duration.Our main results were as follows:1.SYTO9/PI fluorescence staining technology was used to stain different components of Bt XL6 biofilm.Quantitative experiments were performed using a laser confocal microscopy to observe the structural characteristics and formation process of the biofilm at different stages.The results showed that after 18 h of culture,Bt XL6 was still in swimming.After 20 h,the bacteria accumulated to form micro-colonies,which was in the initial stage of matrix formation.With the increase of the culture time,the thickness of the biofilm increased,and the biomass reached the maximum after 34 h of culture.After 56 h,the thickness of the capsule showed a downward trend,the proportion of dead bacteria increased,and the capsule began to decompose.2.Using comparative proteomics methods,the differences in the expression of Bt XL6 between the planktonic state and the biofilm state of total bacterial proteins were studied.The results showed that 885 proteins,including 517 up-regulated proteins and 368 down-regulated ones,were significantly different(ratio≥1.5 fold,p<0.05)between 34 h and 18 h.Between 56 h and 18 h,702 proteins increased and 438 proteins decreased.423 proteins were up-regulated while 433 proteins were down-regulated between 34 h and 20 h.Between 56 h and 20 h,619 proteins were up-regulated,and 522 proteins were down-regulated.Between 56 h and 34 h,581 proteins were up-regulated while 373 proteins were down-regulated.3.Using comparative transcriptomics methods,the differences in the gene transcription of Bt XL6 between the planktonic state and the biofilm state were studied.Using sequencing analysis the comparison group of each group of samples.The results showed that there was no significant difference between 20 h and 18 h;In the 34 h VS 18 h comparison group,335 up-regulated genes and 196 down-regulated genes were detected;In the 56 h VS 18 h comparison group,695 genes were up-regulated and 462 genes were down-regulated.In 34 h VS 20 h,92 genes were up-regulated and 69 genes were down-regulated;In the 56 h VS 20 h comparison group,287 genes were up-regulated and 260 genes down-regulated.In 56 h VS 34 h,13 genes were up-regulated and 8 genes were down-regulated.4.Through the integrated analysis of comparative proteomics and comparative transcriptomics,the key regulatory proteins and the temporal expressed genes involved in Bt biofilm formation and dispersion were studied.The results showed that there were no up-regulated and down-regulated genes at 20 h VS 18 h,while 36 genes were up-regulated and 6 genes were down-regulated at 34 h VS 18 h.49 genes were up-regulated and 14 genes were down-regulated at 56 h VS 18 h.34 h VS 20 h had 2 genes up-regulated and 6 genes were down-regulated.24 genes were up-regulated and 13 genes were up-regulated at 56 h vs 20 h.1 gene was up-regulated at 56 h vs 34 h.The integrated analysis resulted in a dramatic decrease in the number of differential protein/genes at each time points,which greatly facilitated the subsequent study of important regulatory genes.5.The Bt XL6 regulatory pathway and the cross-linking network of biofilm were initially constructed with this study and the reference to previously successfully built biofilm regulatory networks,thereby revealing the mechanism of biofilm formation.According to the literature search,six new key proteins and genes related to Bt XL6 biofilm formation and dispersion have been found in this paper,which lays a solid foundation for the construction of engineering biofilms based on synthetic biology.