| Lysobacter enzymogenes OH11,isolated from rhizosphere soil of pepper,is a novel biocontrol bacterium in 2003. OH11 has a bright future in biocontrol application, exhibited strong antifungal activity against many phytopathogens, as it could produce diverse extracellular chitinase enzymes, HSAF and WAP-8294A2. LeClp is an important global regulator. In previous study, Leclp can control the biosynthesis of chitinase and antimicrobial secondary metabolites, but the mechanism is unknown. In this study we take Lysobacter enzymogenes OH11 as research object and make a deep study in the mechanism of LeClp play a role in regulating of above three biocontrol factors. The following results were obtained.Based on the genome sequence, we constructed the vector for LeClp expression,optimized the induction and purification of protein LeClp conditions and got a large number of purified protein of LeClp. This is a good foundation for related experiments,such as EMSA assay. We examined the combination between protein of LeCIp and c-di-GMP by microscale thermophoresis (MST). These results indicated that LeClp can be combined with c-di-GMP in Lysobacter enzymogenes OH 11 and LeClp is a receptor of c-di-GMP. Its role in the regulation correlated with the content of intracellular c-di-GMP.In our previous study, the Leclp-deletion mutant ?Leclp lost the production of extracellular chitinase, but the mechanism is unknown. chiA was a crucial gene in chitinase biosynthesis. Bioinformatics analysis shown that the promoter of chiA may have LeCIp binding sites, which was confirmed by bacterial one-hybrid assay, EMSA and ChIP-PCR detection method. The global regulator LeClp controls the production of extracellular chitinase through regulating the transcription of chitinase-encoding gene chiA through a direct binding to its promoter region in vitro and in vivo. In the experimental conditions,c-di-GMP does not affect the binding of LeCIp and promoter. This result suggested that LeClp play an essential role in controlling chiA expression did not depend on the content of c-di-GMP. It is important that we find the direct interaction between LeClp and the chiA promoter appears to be a conserved mechanism in diverse chitinase-producing Lysobacter species.pks/nrps was a crucial gene in HSAF biosynthesis. The pks/nrps -deletion mutant?pks/nrps results in an almost deficiency in producing HSAF. Bioinformatics analysis shown that the promoter of pks/nrps may have LeClp binding sites, which was confirmed by bacterial one-hybrid assay and EMSA. These results indicated that the global regulator LeClp controls the biosynthesis of HSAF through a direct binding to pks/nrps promoter region in vitro. In the experimental conditions, c-di-GMP does not affect the binding of LeClp and promoter. This result suggested that LeClp play an essential role in controlling pks/nrps expression did not depend on the content of c-di-GMP. In addition, we suggest that LeClp as downstream factor, mediated the process of LesR (solo LuxR) regulate the biosynthesis of HSAF, through the comparative transcriptome analysis and biochemical test.WAP-8294A2 was a crucial factor in biocontrol process of Lysobacter enzymogenes OH 11 and in our previous study the Leclp-deletion mutant ?Leclp lost the production of WAP-8294A2. We found the promoter of WAP-8294A2 synthetic gene through RT-PCR.The result of bacterial one-hybrid shown that LeClp does not directly regulate the biosynthesis of WAP-8294A2. This mechanism is completerly different from the mechanism of LeClp regulate the HSAF biosynthesis. Using high-throughput ChIP-Seq, we found the downstram gene OH11-1052, which is associated with the biosynthesis of WAP-8294A2. This result was confirmed by bacterial one-hybrid assay and EMSA. These results indicated that LeClp can be directly bind to the promoter of OH11-1052 to regulate the biosynthesis of WAP-8294A2 and the c-di-GMP can not influence the binding of LeClp and the promoter of OH11-1052. |
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