The Molecular Mechanisms Of Biofilm Formation Regulated By Two-Component Regulatory System Resde In Bacillus Amyloliquefaciens SQR9

Efficient root colonization is the precondition of PGPR to exert its beneficial effects in the rhizosphere,and the ability of biofilm formation by PGPR is important to root colonization.Biofilm formation by Bacillus strains is triggered by environmental signals.Oxygen deficiency has been highlighted to induce the biofilm formation of Bacillus,but the specific mechanism of signal recognition is not thoroughly elucidated.Bacillus amyloliquefaciens SQR9,isolated from rhizosphere of a health cucumber plant root in diseased planting region by Jiangsu Provincial Key Lab for Organic Solid Waste Utilization,was proved to be an excellent PGPR strain via its ability to suppress Fusarium oxysporusm.Our previous study found that the two-component regulatory system ResDE was involved in root colonization and biofilm formation,but the involved mechanism is still unclear.In this paper,we try to investigate that how ResDE related to oxygen deficient signal and biofilm formation by gene knock-out,ITC assay,EMSA assay,DNase I footprint assay and BACTH assay.The main results obtained were summarized as follows:1.Mechanism of the two-component regulatory system ResDE involved in regulation of biofilm formation by SQR9 strain under oxygen deficient condition was elucidated.Environmental oxygen deficiency could induce SQR9 biofilm formation.The up-regulated expression of genes related to extracellular matrix synthesis under oxygen deficient condition suggested that extracellular matrix contribute to the enhancement of SQR9 biofilm formation under this condition.Observation of colony structures of SQR9MresD and SQR9MresE strains under various oxygen concentrations and transcriptional analysis of genes related to biofilm formation indicated that ResDE was involved in the oxygen deficiency-induced biofilm formation of SQR9.2.We identified the constitute promoter PreSA and the auto-regulate promoter PresD,both of which are responsible for the regulation of resABCDE and resDE genes expression,respectively.The analysis of promoters proved that PresA and PresD existed in the upstream of resA and resD gene,respectively.The promoter PresA showed strong transcriptional activity while PresD showed weak activity.The increasing GFP intensity of PresD::gfp and expression levels of resD/E genes under oxygen deficient condition indicated that ResDE was induced under this condition.Quantification of the NAD+/NADH ratio and ITC assay suggested that ResE could sense the drop of NAD+/NADH ratio via binding to NAD+ in a PAS-domain dependent manner.3.Observation of mutant strains' colony structures under oxygen deficient condition indicated oxygen deficient signal promoted SQR9 biofilm formation by impairing respiration process.The down-regulated expression of ctaBCDEF(encodes cytochrome caa3),qcrABC(encodes cytochrome bc)and qoxABCD(encodes cytochrome aa3)suggested that ResDE affected the synthesis of cytochrome caa3,cytochrome aa3 and cytochrome be.Electrophoretic mobility shift assay and DNase I footprint assay proved that ResD protein could directly and specifically regulate the expression of ctaBCDEF and qoxABCD operons.4.To figure out how cytochrome complex participates in the process of biofilm formation under oxygen deficient condition,mutants of ctaBCDEF,qoxABCD and qcrABC operons were constructed.Observation of colony structures of SQR9Mcta,SQR9Mqcr and SQR9Mqox under various oxygen concentrations suggested that cytochrome aa3 played an important role in the response of SQR9 to oxygen variation.Transcriptional analysis of genes related to biofilm formation in SQR9Mqox,indicated that cytochrome aa3 was involved in SQR9 biofilm formation.We proved that kinase KinB was also involved in oxygen deficiency-induced biofilm formation process by gene knock-out and colony structure observation under various oxygen conditions.BATCH assay proved that cytochrome aa3 interacted with KinB in a protein-protein interaction manner.All these results showed indicated that cytochrome aa3 regulated biofilm formation via a KinB-Spo0A pathway.5.Comparation of biofilm formation ability of SQR9,PresD-resD,P43-resD and pNW33N-P43-resD strains showed that the up-regulated expression of resD within a certain limit could promote the biofilm formation,but the excessive expression of resD would impair the biofilm formation.We thus quantified the production of eDNA and measured the ratio of cell lysis of PresD-resD,P43-resD and pNW33N-P43-resD compared with SQR9 and the results suggested that up-regulation of resD impaired the stability of cell wall and membrane structures,causing cell lysis and eDNA releasing.We suggested that lytA,IytB,IytC,yycE,xylA,xylB genes migth be regulated by ResD based on the transcriptional analysis of petidoglycan hydrolases in SQR9.These results suggested that ResDE could also participate in the process of SQR9 biofilm formation by regulating the activities of petidoglycan hydrolases.Based on our previous study,here we conclude that the two-component regulatory system ResDE recognizes the signal of environmental oxygen deficiency and thus regulates biofilm formation of SQR9 via electron transport chain.In addition,ResDE also participates in the biofilm formation process of SQR9 by regulating the activity of petidoglycan hydrolases.All these results could provide theoretical instructions for improving the root colonization and agricultural application of B.amyloliquefacien SQR9.