Mechanism Research Of DF(Diffusible Factor)Quorum Sensing System In The Biosynthesis Of Antimicrobial Secondary Metabolites Hsaf In Lysobacter Enzymogenes OH11

Lysobacter enzymogenes OH11 classified to the Xanthomonadaceae family and isolated from rhizosphere soil of pepper,is a ubiquitous environmental bacterium emerging as a potentially novel biological control agent.The HSAF(Heat-Stable Antifungal Factor)it produces can inhibit the growth of a variety of plant pathogenic fungi and has a bright future in biocontrol application.DF(Diffusible Factor)is a type of key quorum sensing system in bacterial.Previous studies have shown that DF played positive effects in modulating HSAF biosynthesis,but the mechanism is not clear.In this study,we take Lysobacter enzymogenes OH 11 as the research object and have an employed study in the mechanism of DF playing a role in regulating of HSAF.The results are as follows:We have confirmed that the key genes involved in shikimate pathway are existing conservedly in Lysobacter enzymogenes OH11,and construct the aroA gene knock-out mutant.The aroA gene played positively role in the biosynthesis of yellow pigment and DF.At the same time,the DF is 3-hydroxy-benzoic acid(3-HBA)and 4-hydroxy benzoic acid(4-HBA).Furthermore,the aroA gene played positively role in the biosynthesis of HSAF,which revealed the shikimate pathway regulating the biosynthesis of HSAF through DF.Previous study has discovered that the mutant △lenB2 destroyed the production of HSAF,revealing LenB2 playing role in the biosynthesis of HSAF,but the mechanisms of molecular genetics and biological chemistry are unknown.The research verified that LenB2 catalyzes the shikimate pathway’s end-products chorismate to generate DF by carrying out genetic assays and biochemical assays,illustrating that LenB2 is the crucial enzyme of producing DF.Adding DF in the AaroA、△lenB2、△aroAAlenBcin vitro and introducing the ubiC’gene related to catalyzing chorismate to 4-HBA and cuv10 gene related to catalyzing chorismate to 3-HBA into the △lenB2 in vivo confirmed that 4-HBA regulated the biosynthesis of HSAF but not 3-HBA.It is important that just 0.5μM 4-HBA can resume the HSAF production of AlenB2,revealling that 4-HBA act as a signal to regulate the biosynthesis of HSAF.Bioinfonnatics predicted LysRLe is the receptor of 4-HBA,and △lysRLe didn’t produce HSAF,the mechanism of lysRLe regulating prodution of HSAF is that LysRLe directly bound the promoter of key gene IafB involved in biosynthesis of HSAF.Microscale Thermophoresis assay indicated LysRLe directly bound 4-HBA.4-HBA enhanced the affinity of LysRLe binding to lafB’s promoter by using electrophoretic mobility shift assay,showing that 4-HBA regulating the biosynthesis of HSAF is through LysRLe.Identified a factor marRLe regulated by LenB2 through analyzing △lenB2 DNA microarray,and marRLe regulated the biosynthesis of HSAF.marRLe is downstream of lenB2 by carrying out genetic assay.One-hybrid assay and electrophoretic mobility shift assay illustrated that MarRLe directly bound lafB’s promoter resulting in regulating the biosynthesis of HSAF,which was not dependent on 4-HBA Morever,LysRLe not only directly bound lafB’s promoter to regulate biosynthesis of HSAF but also directly bound marRLe’s promoter to regulate the expression of marRLe that also directly bound IafB’s promoter,and LysRLe didn’t interact with MarRLe on protein,revealling two styles related to modulating the biosynthesis of HSAF.