Identification,Functional And Mechanistic Investigation Of CdgL,a Binding Protein Of The Nucleotide Second Messenger C-di-GMP In Lysobacter Enzymogenes OH11

Lysobacter enzymegenes OH 11 is a gram-negative bacterium with biocontrol effect in rhizosphere soil of pepper,which belongs to the group of y-proteobacteria,Xanthomonae Genus.Strain OH 11 seeks habitat-associated pathogenic fungi and oomycetes through twitching motility(TM)mediated by type Ⅳ pilus,and then suppresses their growth through exogenous antibacterial HSAF to achieve the purpose of biological control.In the previous work,it was found that the second messenger of nucleic acid in the strain OH11,cyclic diguanylate(c-di-GMP),could inhibit HASF synthesis after increasing the concentration,and identified the transcription factor Clp as a receptor of c-di-GMP protein.The binding of c-di-GMP will inhibit the ability of Clp to bind to the promoter region of the HSAF synthesis gene cluster,inhibit the expression of HSAF synthesis genes,and then inhibit the synthesis of HSAF.At the same time,the group also identified a diffusible small molecule chemical signal 4-hydroxybenzoic acid(4-HBA)from the OH 11 culture supernatant and found that it positively regulates HSAF synthesis via its receptor transcription factor LysR.The synthesis of 4-HBA in strain OH 11 is responsible for the oxidase LenB2.LenB2 synthesizes 4-HBA and its structural analog,3-hydroxybenzoic acid(3-HBA),by using the branch product of the shikimic acid pathway as a substrate.Unlike 4-HBA,3-HBA is a precursor to bacterial yellow pigment synthesis,but does not affect HSAF synthesis.This project aims to discover a new c-di-GMP receptor protein in Lysobacter enzymegenes OH11,study its function,and explore the possible internal connection between it and the 4-HBA signaling pathway.The main findings are as follows:1.YajQ is a newly discovered c-di-GMP binding protein in Xanthomonas campestris,which is involved in regulating the pathogenicity of the plant pathogenic bacteria and its flagella-mediated motility.By sequence alignment,this study identified a YajQ homologue Le2538 from the Lysobacter enzymegenes OH 11 genome and named it CdgL(c-di-GMP receptor interacting with LysR).CdgL has a 78%amino acid similarity to YajQ of Xanthomonas campestris.CdgL-GST fusion protein was obtained using prokaryotic expression technology.Through Microscale thermophoresis technology,it was found that the CdgL-GST fusion protein can specifically bind to c-di-GMP,and the affinity constant reached 14.62.Through genetic studies of deletion mutations,complementarities,and other phenotypic analyses of CdgL-encoding genes,it was found that CdgL can positively regulate HSAF synthesis and Twitching Motility.In order to dig deeper into the biological functions controlled by CdgL,this study conducted a transcriptomics study and found that CdgL can control the transcription and expression of 373 genes,among them,the key genes of HSAF synthesis gene cluster and type Ⅳ pili synthesis are in the range of CdgL down-regulated genes,which is consistent with the results of CdgL controlling HSAF synthesis and Ⅳ pili mediated Twitching Motility.According to transcriptome data,it was further found that CdgL positively controls the transcription expression of lenB2,revealing that CdgL may be involved in the synthesis of yellow pigment,and was confirmed through research.The above results reveal that CdgL is a newly discovered c-di-GMP receptor protein in Lysobacter enzymegenes OH11,and can regulate the formation of two biocontrol factors of strain OH1 1-the antibacterial substance HSAF and Twitching Motility.2.Through bacterial two-hybrid,pull-down in vitro,and MST techniques,it was found that CdgL can directly interact with 4-HBA receptor protein LysR to form a specific CdgL-LysR protein complex.Through the formation of this complex,CdgL can enhance the ability of LysR to bind to the promoter region of the HSAF synthesis gene cluster,enhance the expression of HSAF synthesis genes,and then promote HSAF synthesis.The physiological concentration of c-di-GMP combined with CdgL or 4-HBA combined with LysR will destroy the formation of the CdgL-LysR complex,and release CdgL from the three CdgL-LysR-DNA complexes,which will cause the ability of LysR binding to the HSAF synthetic gene cluster is reduced,reducing the expression of HSAF synthesis genes,thereby inhibiting HSAF synthesis.These results indicate that c-di-GMP and 4-HBA can simultaneously "attack" the protein complex composed of their respective receptors to precisely control HSAF synthesis,and based on this,the intrinsic communication mechanism between c-di-GMP and 4-HBA signaling pathway mediated by the CdgL-LysR protein complex was established.3.In vitro MST technology,this study found that CdgL can interact with RpoN,a Sigma factor in Lysobacter enzymegenes,but the function of RpoN is unknown.Through genetic studies such as mutation and complementation combined with phenotypic determination,it was found that,similar to CdgL,RpoN can also positively regulate the formation of twitching motility.Genomic scanning revealed 28 Sigma factors in Lysobacter enzymegenes OH 11,but only RpoN controls lame movement,revealing that CdgL interacts with RpoN as one of the molecular mechanisms to regulate twitching motility.In the previous work,the research group found that PilA,a key structural protein of type Ⅳ pili,and hybrid two-component system ChpA,are necessary factors for the twitching motility of the Lysobacter enzymegenes OH11.Through bioinformatics analysis,this study found that RpoN binding sites existed in the promoter region of ChpA coding genes,and confirmed by gel block electrophoresis.Through quantitative detection of gene expression,binding site truncation and site-directed mutations,this study further revealed that RpoN directly promotes the transcriptional expression of ChpA coding gene by binding its promoters directly to the regulation of type Ⅳ pili mediated twitching motility.In summary,this study found a new c-di-GMP receptor protein,CdgL,from the Lysobacter enzymegenes OH11,and revealed its molecular mechanism that mediates c-di-GMP signaling pathways to regulate HSAF synthesis.A network of "CdgL-LysR"protein complex-mediated c-di-GMP-4-HBA synergistically regulates HSAF synthesis.At the same time,this study revealed the molecular mechanism of CdgL to regulate twitching motility through interaction with RpoN.These new basic theoretical findings have laid a theoretical foundation for the subsequent improvement of HSAF yield through molecular improvement of the CdgL-mediated signal network,the twitching motility of strain OH11,the predation ability of strain OH 11 on pathogenic fungal Oomycetes,and the improvement of its biocontrol effect.