Regulation Of FtsEX-CwlO And The Involvement Of Cannibalism In Biofilm Formation Of Plant Beneficial Rhizobacterium Bacillus Amyloliquefaciens SQR9

In recent years,with the extensive use of chemical fertilizer,the quality of soil has declined.The microbial organic fertilizers,which are safe,efficient and environmentally friendly,show great prospect in agricultural application.Bacillus amyloliquefaciens SQR9,isolated from healthy cucumber rhizosphere by our laboratory,is a plant beneficial rhizobacterium.It can form robust and highly structured biofilms and colonize on the root surface.SQR9 has been widely used in microbial organic fertilizers because it has plant growth-promoting and biocontrol function.The widespread recognition that biofilm formation by plant-beneficial bacteria is crucial for their root colonization and function has resulted in increased interest in understanding molecular mechanisms related to biofilm formation.The production of the matrix is controlled by many genetic pathways and involves multiple transcription factors,which have been studied widely in domesticated strain Bacillus subtilis.Despite the intensive studies of the spo0A pathway for controlling Bacillus biofilm formation,regulation factors upstream of the master regulator Spo0A has not been well investigated.On the other hand,there is a mechanism called cannibalism in B.subtilis.Cannibalism means cannibal cells can lyse a fraction of their sensitive siblings,which can enhance biofilm development.However,it has not been reported that whether cannibalism existed in other PGPR.We screened the mutants in a transposon library which have disruptions of most of the uncharacterized genes in the SQR9 genome.Through mapping of transposon insertion sites and gene knockout,we identified a novel gene involved in biofilm formation and root colonization.Then we investigated how this gene regulates SQR9 biofilm formation.On the other hand,we found that the novel natural secondary metabolites produced by SQR9 can induce cannibalism in biofilm formation.What's more,we studied the cannibalism mechanism involved in SQR9 biofilm formation.The main results obstained in this research were summarized as follows.1.We took advantage of a transposon library and performed the transposon insertion sites analysis as well as gene knockout.In this way,we selected one mutant failure to form wild-type pellicles from 3000 mutants.Then we identified the gene ftsE(encoding the ATP-binding protein of a FtsEX ABC transporter)conferring this unusual phenotype.The mutant AftsE was constructed by gene knockout.The mutant AftsE exhibited growth rate similar to that of SQR9,but its biofilm is defective after incubation for 20 h in MSgg medium.The ftsEX gene fragment was cloned into the B.subtilis-E.coli shuttle vector pNW33N,and we obtained the complementation strain(AftsE pNW33N-ftsEX)successfully.Based on qualitative and quantitative analysis,the wild-type phenotype was restored in strain AftsE pNW33N-ftsEX.In the end,we fused the protein FtsEX with GFP and we found that the protein FtsEX is located on the cell membrane.2.Previous work showed that the FtsEX complex controls the activity of the major autolysin Cw1O.Overexpressed gene cwlO in SQR9 could lead to high autolysin activity and cell damage,which suggested that Cw1O does have autolysin activity in SQR9.Then we constructed the ?cwlO mutant.Through biofilm analysis,the ?cwlO mutant forms pellicles that are thin and flat similar to the pellicles of the AftsE mutant.Additionally,the cwlO mutation could be complemented with a copy of the wild-type gene that had been inserted into the AcwlO chromosome at the amyE site;this complementation resulted in proper pellicle formation,indicating that the biofilm formation of the AcwlO mutant was only caused by the loss of Cw1O function.To further investigate whether the strains SQR9,?ftsE,and AcwlO differ in their ability to form biofilms on plant root surfaces,their colonization on roots of cucumber were compared.The number of roots colonized by mutants AftsE and AcwlO were similar but significantly lower than that of SQR9.These observations indicate that the involvement of FtsEX in biofilm formation depends on its regulation of Cw1O.In the end,cells of SQR9 and the ?cwlO mutant grown under biofilm-forming conditions were microscopically examined.Moreover,extracellular DNA content in cultures of the ?cwlO mutant and SQR9 were evaluated.The results showed that Cw1O is not essential for cell separation or extracellular DNA release.Taken together,these results suggest that the loss of autolysin activity of the AcwlO mutant is not the main reason for biofilm defect.In addition,we also explored the effect of some other autolysins mutation on SQR9 biofilm formation,but we didn't discover an autolysin that could affect biofilm formation.3.To investigate the molecular mechanisms of biofilm formation affected by FtsEX-Cw1O,we constructed the ?cwlOAftsE double mutant.We observed that the mutants?ftsE,?cwlO and AcwlO?ftsE share the similar phenotype on biofilm formation and colony architectures.And all these mutations cause a delay in pellicle development.Subsequently,we used scanning electron microscopy(SEM)to visualize the extracellular matrix formed by the wild-type and mutant strains.Moreover,we tested the extracellular polysaccharides produced by the wild-type and mutant strains.Results showed that the matrix formed by these mutants included less extracellular material including extracellular polysaccharides.To investigate which component in the biofilm matrix was affected in these mutants,the transcription levels of biofilm related genes ? in AftsE,?cwlO and ?cwlO?ftsE mutants were analyzed and compared with the wild-type strian SQR9.The results showed that all three matrix genes,epsD,tapA and bslA,were down-regulated in the mutants during biofilm formation.This effect can be attributed to their regulation of extracellular matrix production through the spoOA pathway,since the transcription of the spoOA cascade genes were altered.In the end,we examined the effects of membrane-bound sensor histidine kinases KinB,KinC and KinD on biofilm development,the results showed that only KinB and KinD are involved in this progression.To investigate whether physical interactions exist between FtsEX/Cw1O and kinases KinB/KinD,we performed the bacterial two-hybrid analysis.Cw1O was shown to exhibit physically interaction with KinB and KinD.So Cw1O influences biofilm formation through the spoOA pathway by possibly interacting with the kinases KinB and KinD.4.Through visualization of SQR9 biofilm by scanning electron microscopy(SEM),we discovered that there are some lytic bacteria in SQR9 biofilm,which suggested a possibility that SQR9 has a social behavior named cannibalism.The level of cell lysis and extracellular DNA was measured and compared in SQR9(wild type),the mutant SQR9MA(self-immunity deficient mutant)and the mutant SQR9MV(toxin deficient mutant)during biofilm formation,then the possibility of the cannibalism phenomenon in SQR9 biofilm was initially identified.To investigate the mechanism of self-immunity induced by toxin Bacillunoic acid A/B,mapping of the different lengths of bnaA-B promoter region by 5'deletion and fusion to gfp gene was performed.In this way,we found the promoter region and predicted its transcription start site,-10 box and-35 box by software.Then the reporter plasmid was introduced into SQR9 and the mutant SQR9MV to construct the strain SQR9-PbnaA-GFP and SQR9MV-PbnaA-GFP,respectively.Next,the induction experiment by fermentation liquid of two constructed strain was conducted and observed.The result indicated that the expression of the bnaA-B promoter was induced by toxin Bacillunoic acid A/B.Subsequently,the different expression of bnaA-B promoter induced by Bacillunoic acid A/B during biofilm formation in two constructed strain was observed and compared.The result indicated that cannibalism existed in SQR9 biofilm formation.In the end,we measured and compared the biofilm formed by SQR9(wild type),the mutant SQR9MA(self-immunity deficient mutant)and the mutant SQR9MV(toxin deficient mutant).The result indicated that cannibalism can induce SQR9 biofilm formation.And biofilm formation would be defective in the absence of toxin Bacillunoic acid A/B.This study revealed that FtsEX-CwlO and cannibalism play a so far undiscovered regulatory role during biofilm formation of SQR9,which may help to enhance root colonization and plant-beneficial function of SQR9 and other beneficial rhizobactreria as well.