Mechanism Research Of Transcriptional Regulator LtetR In The Biosynthesis Of Antimicrobial Secondary Metabolites HSAF In Lysobacter Enzymogenes

Lysobacter enzymogenes OH11,isolated from rhizosphere soil of pepper,is a novel biocontrol bacterium.It exhibits gliding motility(Twitching motility,TM),without flagellum,and has high G+C%content in the gemome.This kind of bacterial can produce various extracellular enzymes and small molecule antibacterial natural products which have the broad-spectrum antimicrobial activity against kinds of phytopathogens.HSAF(Heat Stable Antifugnal Factor)was isolated and identified as a secondary metabolites which has broad-spectrum activity against fungi and Oomycetes,has a novel structure and mode of action.Although our lab has clarified the biosynthetic pathway of the HSAF in Lysobacter enzymogenes,the the regulatory mechanisms of HSAF synthesis is still largely unclear.Transcription factors(TFs)are widely distributed in bacteria and are a class of protein molecules that bind to specific sequences upstream of the target gene 5' to ensure that the target gene is expressing at a specific time at a specific time and space,which are widely distributed in bacteria.The interaction between transcription factors and target DNA has widespread biological signficance in regulatory of gene expression.In this study,we take Lysobacter enzymogenes strain OH 11 which can produce antifungal antibiotics of HSAF,studied the mechanism of biosynthesis of HSAF by transcription factors in Lysobacter enzymogenes strain OH11.In the genome of the Lysobacter enzymogenes OH 11 363 transcription factors was found by gene annotation.These TFs were further divided into 4 functional subgroups based on their functional domain category.In the following IV contents,the TFs containing the HTH(Helix-Turn-Helix)DNA binding domain were selected for generating a Bacterial one-hybrid system library and possessing 87 different TFs.This library was further for screening potential candidate TFs which would direct binding to HSAF promoter region.We identified 10 TFs that directly bind to PHSAF(the promoter region of the HSAF biosynthealsosis operon)in our test system by B1H.These 10 TFs were in-frame deleted individually in the wide type of OH11.All the mutants were selected for testing their HSAF yield,and we found that HSAF production was significantly raised in only one mutant(AletR).Subsequent bacterial one-hybrid system and Electrophoretic Mobility Shift Assay(EMSA)also demonstrated that LetR can bind directly bind to the PHSAF region.DNA truncation assays further identified a core region of PHSAF responsible for LetR binding.In-frame deletion of letR in wild-type OH 11 significantly increased HSAF levels and decreased the transcription of the key biosynthetic gene pks/nrps(lafB),while overexpression of letR in the wild-type background remarkably reduced HSAF levels and decreased the transcription of pks/nrps(lafB).Together,this study identified a new regulator involved in HSAF biosynthesis,also generated a higher HSAF-producing strain(?letR),which represents a robust step in promoting HSAF in pharmaceutical and biological control purposes.