| Lysobacter enzymogenes OH11 is an important agricultural Gram-negative bacteria isolated from the rhizosphere soil of pepper,which can produce a variety of extracellular enzymes,including chitinase,protease,cellulose,β-1,3-glucanases,which can degrade cell wall components of different phytopathogens,are a kind of biocontrol bacteria with broad application prospects.At the same time,the bacteria can also synthesize and excrete a broad-spectrum antifungal and oomycete secondary metabolite HSAF,which acts on the biosynthesis of filamentous fungi sphingolipid compounds,thereby inhibiting the growth of pathogenic fungi.In recent years,related studies have shown that Lysozyme-producing bacteria also can rely on bacterial type Ⅳ fimbriae(T4P)adsorbed on the fungal hyphae,so that mycelia digestion to produce biocontrol effect so by T4P-mediated lameness movement also used as an important indicator of the ability of L.enzyme-prodncing bacilli.In this paper,L enzymogenes OH11 was used as the research object to explore the mechanism of the bacteria’s production of clonic movement.In L.enzymogenes,Clp acts as a global regulator that regulates the biosynthesis of chitinase,secondary metabolites,and the motility of the bacteria.After previous research in our laboratory,the first two regulatory mechanisms have made corresponding progress,but the mechanism of twitching motility is unknown.Firstly this article through the gel electrophoresis(EMSA)test in vitro,founding that Clp can be directly integrated in the promoter region of the gene encoding PilA of type Ⅳ pilus synthesis protein.Further through promoter truncation and EMSA techniques,the binding region of Clp was mapped to a 41 bp DNA sequence.Through bioinformatics analysis,the potential binding site of Clp,GTG**********CAC,was found in this 41-bp DNA sequence.Through genetic and biochemical studies such as quantitative gene expression,constitutive promoter replacement,and Western blotting hybridization,it was found that this 41 bp sequence was indeed a binding site for Clp.Using the Clp binding site found in the pilA promoter region as a bait,scanning was performed within the entire genome of OH11 and 222 gene promoter regions were found to contain similar binding sites.From this we found that there was also a Clp binding site in the promoter region of the synthetic gene cluster PilMNOPQ responsible for the assembly of type Ⅳ pilus in the periplasmic space.Through in vitro EMSA and in vivo gene expression analysis,it was found that Clp be directly integrated into the promoter region of pilM,thereby positively controlling the transcription of this gene cluster.These studies indicate that Clp in L enzymogenes OH11 controls cell lameness mediated by type IV pilus through a dual transcriptional regulatory mechanism,and this regulation is relatively conserved among many Lysobacter species.Our previous experiments found that there were 26 proteins associated with the c-di-GMP anabolic metabolism in the OH11-producing enzyme,Le2996(lctM)containing the degenerate GGDEF domain has been lost after overexpression.As a result of the twitching motility,this paper has studied the molecular mechanism behind this phenomenon.Bioinformatics analysis showed that the N-terminus of LctM was integrated in the bacterial intima and contained six transmembrane regions.The C-terminus of LctM was located in the cytoplasm and contained a degenerate GGDEF domain,indicating that this protein can’t synthesize c-di-GMP.In order to explore the mechanism of LctM regulation of TM,We used LctM as a decoy to perform a one-to-one bacterial two-hybrid screening on 19 Pil proteins that can control the synthesis of type Ⅳ pilus and found that PilO,a transmembrane protein,was specific to LctM interaction.This article initially revealed the global regulatory factors Clp and LctM containing degenerate GGDEF domain to regulate the movement of lameness,to enhance the ability of TM through genetic improvement and to better colonize the plant or fungal hyphae foundation. |
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