The Molecular Mechanisms Of The Effects Of Quorum-sensing Signal AI-2 On The Motility And Biofilm Formation In Bacillus Amyloliquefaciens SQR9

Plant growth-promoting rhizobacteria(PGPRs)are beneficial microorganisms which can survive and colonize on rhizosphere and have the ability to promote growth or prevent soil-borne diseases,such as Bacillus spp.,Streptomyces spp.,Pseudomonas spp.,Burkholcderia spp.and so on.In recent years,PGPRs have been widely used in agricultural production as microbial fertilizer because of its environment-friendly,safe,non-toxic and other advantages.And they have achieved good results in promoting plant growth and the control of soil-borne diseases.Bioilm formation and colonization of microbes on plant rhizosphere are affected by many factors,including bacterial flagella/motility,chemotaxis,polysaccharides,and plant root exudates et al..These factors are related to the regulation of a variety of signal substances,such as extracellular signals,signal substances of various bacteria-plant interaction in the root exudates,as well as a variety of regulatory signals produced by the cell itself,such as cell self-generated quorum sensing signal(quorum-sensing,QS)substances.Bacillus amyloliquefaciens SQR9(SQR9),isolated and identified by our laboratory,was proved to be an outstanding plant growth-promoting rhizobacterium(PGPR)strain.In this study,we want to know the molecular mechanisms of the effects of quorum-sensing signal AI-2 on the motility and biofilm formation in Bacillus amyloliquefaciens SQR9.First,we got the AI-2-luxS mutant(SQR9-A/uxiS)by gene knockout.And compared the biofilm formation and motility(swimming/swarming)with the wild-type SQR9 using crystal violet staining methord.Second,the extracellular polysaccharides of SQR9-?luxS were tested by sulfuric acid anthrone titration method.Then,the wild-type,mutant and complementary strains were tagged by green fluorescent protein.Plate counts and a laser confocal fluorescence microscope were used to observe the rhizosphere colonization in situ of cucumber and tomato.Furthermore,transcriptome sequencing of the cells of wild-type and mutants at different time points(8h,18h,32h and 60h)were conducted and analyzed.Based on the transcriptomic data,the biofilm formation and other regulatory pathways were deduced,for example,biofilm-related genes or motility-related genes.Finally,we predicted the AI-2 receptor proteins in SQR9 according to the known related information about AI-2 receptor proteins,and a series of verification experiments that we are doing.As a conclusion,the main results of this study are as follows:1.We got the AI-2-luxS mutant(SQR9-?luxS)by gene knockout,and then complemented successfully,obtained the complementary strain C-AluxS;2.Biofilms formation,motility and EPS production of SQR9-?luxS and wild-type SQR9 were measured and compared.There was a significant increase of biofilm formation in mutant SQR9-?luxS,and both motility and extracellular polysaccharide also obviously increased;3.The wild-type,SQR9-?luxS and C-?luxS of SQR9 were tagged with GFP,and got SQR9-gfp,SQR9-?luxS-gfp and C?luxS-gfp respectively.The GFP-tagged strains were inoculated into the sterile MS solution with cucumber and tomato.The bacteria numbers of plates from cucumber roots which were inoculated with SQR9-?luxS-gfp was 3.05*106/g,this was more than the inoculated plants by SQR9-gfp(1.6*106/g)and C-AluxS-gfp(1.85*106/g).With the increase of the added AI-2 concentration,the number of colonizing bacteria reduced significantly.Confocal obervations further verified the plates counting.4.The transcriptome data indicated that the significant gene expression differences between SQR9-?luxS and wild-type mainly showed in the logarithmic growth stage both on the LB and M9-mannitol medium.And these different genes included the genes such as biofilm formation,flagellar synthesis(motility)and sugar transporter protein,especially the data indicated that AI-2 also had an important regulatory role in some secondary metabolic pathways of SQR9.A large number of genes related to sugar transportation were much higher expression in luxS mutant than the wild type of SQR9,and the levels of the gene expressions in the luxS mutant were at least four times than those of wild type,which suggesting that AI-2,as a signal molecule,also had an important regulatory role in cell glucose metabolism.5.Foxur possible AI-2 receptor proteins were found in SQR9 based on the structures,physicochemical properties and gene cluster characteristics of the known AI-2 receptor proteins in the Gram-negative strains.