| Bacteria have ability to respond quickly to changes in external environment so that they can survive better.Chemotaxis(Che)is a major form of behavior in which bacteria move from perception of environmental changes to response to flagella movements that are beneficial to their survival and away from harmful environments.Comparative genomics revealed that the genomes of different bacteria encode one or more variant Che systems.These chemotactic variation systems include most components of Che system,but their functions have undergone significant differentiation.They not only control the chemotaxis of bacteria,but also differentiate into a variety of new biological functions to control bacterial biofilm formation and pathogenicity.One of the typical chemotactic variation systems is the Wspsystem from animal pathogenic bacteria-Pseudomonas aeruginosa,which contains five conserved homologous proteins of Che system : MCP(WspA),Che R(WspC),Che B(WspF),Che W(WspB and WspD)and Che A(WspE).When flagellated P.aeruginosa senses the unknown signal on the solid surface,WspA methylation is accelerated by activating WspC methyltransferase activity or inhibiting WspF methylesterase activity,thereby inducing WspE phosphorylation.Phosphorylated WspE transfers the phosphate group to WspR(an intracellular second messenger c-di-GMP synthetase with a REC domain at the N-terminal receiving phosphate group and a GGEEF active domain at the C-terminal responsible for the synthesis of c-di-GMP),and then phosphorylates WspR to improve its synthase activity and promote the increase of intracellular c-di-GMP concentration.The increase of intracellular c-di-GMP content further promotes the formation of c-di-GMP and receptor Fle Q complex,in which Fle Q is a transcription factor regulating flagellar synthesisrelated gene expression.The combination with c-di-GMP will weaken the ability of Fle Q to bind to the promoter region of flagellar synthesis-related genes,thereby reducing the transcription of flagellar synthesis-related genes,leading to the weakening of bacterial motility and inducing the formation of aggregation state(biofilm),thus realizing the Wspsystem to regulate the transformation of P.aeruginosa from movement to aggregation lifestyle through c-di-GMP and promoting its infection.At present,the regulation function of Wspsystem in pathogenic bacteria has been studied thoroughly,but it has not been reported in biocontrol bacteria.Lysobacter enzymogenes OH11 is a non-flagellated biocontrol Gram-negative bacterium.After losing flagella,OH11 can move on the solid surface with type IV pilus(T4P).As a biocontrol bacterium,OH11 can prey on fungi in the natural environment.When sensing the presence of surrounding fungi,OH11 approaches the fungi with T4 P,and then forms biofilms that colonize the hyphae.Subsequently,OH11 secretes heat-stable antifungal factor HSAF to inhibit mycelial growth,and then excretes extracellular hydrolytic enzymes such as chitin to break fungal hyphae to achieve the purpose of nutrition.In this thesis,OH11 was taken as the object to explore the functional evolution of chemotaxis variation system Wspin nonflagellated biocontrol bacteria and carry out in-depth mechanism analysis.The main research progress is as follows :(1)We found that OH11 encoded a complete Wspsystem by bioinformatics analysis.Through genetic studies on deletion mutation and complementation of gene clusters of Wspsystem with phenotypic analysis,we found that WspF could positively regulate HSAF biosynthesis and negatively regulate biofilm formation.The above results revealed that the Wspsystem in OH11 not only retained the regulatory function of biofilm formation,but also evolved a new function to regulate the biosynthesis of HSAF.(2)The quantitative detection of HSAF of the mutant strain with the key phosphorylation sites and enzyme activity sites of WspR,the core component of Wspsystem,showed that the system regulated the biosynthesis of HSAF by WspR-mediated c-di-GMP signaling pathway.Because OH11 loses flagellum in evolution,its genome lacks Fle Q homologous protein,suggesting that Wspsystem may regulate HSAF biosynthesis through new c-di-GMP receptors.Clp and Cdg L are two identified c-di-GMP receptors and positive regulate the biosynthesis of HSAF in OH11.Therefore,we speculated that WspR may direct interact with them to transmit c-di-GMP and then regulate the biosynthesis of HSAF.Through bacterial two-hybrid,pull down and MST,we found that WspR could specifically interact with Cdg L,but not Clp.Further truncation of the domain of WspR showed that the N-terminal REC domain was the key region for interaction with Cdg L.Since the REC domain contains phosphorylated key amino acid residues,we speculated that the phosphorylation of WspR may affect the interaction with Cdg L.In order to verify this hypothesis,MST assay was performed to find that WspR phosphorylation weakened the interaction with Cdg L,while cdi-GMP did not affect the interaction between WspR and Cdg L.Recent studies shown that Cdg L is not a transcription factor,but can interact with a transcription factor Lys R to enhance the ability of Lys R to bind to the promoter region of the key gene cluster in HSAF biosynthesis,thereby promoting the biosynthesis of HSAF.Based on the above results,this thesis revealed the molecular mechanism of co-evolution of Wspsystem function and regulation mode in non-flagellated L.enzymogenes OH11.(3)In addition to regulating the biosynthesis of HSAF,we found that Wspsystem retained the function of regulating biofilm formation.Through the detection of biofilm formation of Wspdeletion mutants and key site mutations,we found that Wspsystem regulated biofilm formation by WspR-mediated c-di-GMP signalling pathway.In order to identify whether other c-di-GMP metabolic enzymes in OH11 are involved in the regulation of biofilm formation,we detected the biofilm formation of reported synthase Lch D,degradation enzymes Lch P and RpfG,and found that RpfG regulates biofilm formation.As a c-di-GMP degrading enzyme,RpfG belongs to DSF-type quorum sensing system(QS),which is responsible for degrading intracellular c-di-GMP and regulating the corresponding biological functions.We found that RpfG regulates biofilm formation in a manner independent of its own enzyme activity,suggesting that QS may rely on Wspsystem to regulate biofilm formation.The interaction between WspR and RpfG was found by bacterial two-hybrid,pull down and MST,and the interaction between the GGEEF domain of WspR and the HD-GYP domain of RpfG was further found.In vitro biochemical enzyme activity assay revealed that the interaction between RpfG and WspR could inhibit the activity of WspR synthetase.Furthermore,we found that Clp deletion promoted the biofilm formation,while Cdg L deletion did not affect the biofilm formation.Accordingly,this thesis revealed that molecular mechanism of Wspsystem and QS synergistically on regulating biofilm formation in L.enzymogenes OH11.In summary,this thesis elucidated the molecular mechanism of Wsp-mediated c-di-GMP signaling pathway regulating HSAF biosynthesis in non-flagellated L.enzymogenes OH11,and proposed a molecular network of c-di-GMP specific transmission mediated by ’WspRCdg L’ compound.This thesis revealed the molecular mechanism of synergistic regulation of biofilm formation by Wspsystem and DSF-type quorum sensing system.These findings provide a theoretical basis for further genetic improvement of c-di-GMP signaling network mediated by Wspsystem to improve HSAF production and enhance the colonization ability of L.enzymogenes OH11. |
