Identification And Functional Characterization Of Transcription Factors Interacted With The Global Regulator Clp Protein In Lysobacter Enzymogenes

Lysobacter enzymogenes OH11 is a broad application prospect.biocontrol bacterium which was isolated from rhizosphere soil of pepper by our laboratory.OH11 which displays strong inhibition against kinds of Plant Pathogenic Fungi,Oomycetes and gram-positive bacteria,can produce heat stable antifungal factor HSAF,a variety of extracellular hydrolytic enzymes and antibacterial secondary products.Clp(cAMP-receptor like protein)is a transcriptional regulatory protein,which belongs to the CRP(cyclic AMP receptor protein)protein family.According to our previous studies,absence of clp resulted in the inability of HSAF to synthesize in Lysobacter enzymogenes.Further studies reveal that clp mutations can significantly reduce the transcription of HSAF biosynthetic gene pks/nrps expression.We found that Clp could regulate HSAF biosynthesis by directly binding to the promoter region of the pks/nrps gene.By screening transcription factors interacting with Clp protein in Lysobacter enzymogenes,we find the target transcription factors to reveal the molecular mechanism of Clp regulating HSAF synthesis.On this basis,we investigated how the target transcription factor can regulate HSAF biosynthesis by interacting with Clp protein.Based on the Lysobacter enzymogenes OH11 transcription factor pTRG library,we through the bacterial two hybrid system to screen for 14 transcription factors interacted with Clp.Moreover,we knockouted 14 target transcription factors and obtained deletion mutants.By detecting the production of HSAF,antifungal activity of pathogenic fungi,TM,and the extracellular enzyme,we identified that the level of Le1703 mutant's HSAF is significantly lower than that of OH11.Through NCBI analysis,we name Le1703 as LysRLe.These results suggested that LysRLe is involved in the regulation of HSAF biosynthesis.A large number of purified LysRLe proteins were obtained by constructing prokaryotic expression vector of LysRLe protein.EMSA and bacterial single hybrid experiments showed that LysRLe,could directly regulate the biosynthesis of HSAF in the promoter region of pks/nrps gene.In Lysobacter enzymogenes OH11,Clp regulates the biosynthesis of HSAF by directly binding to the promoter region of pks/nrps gene.LysRLe can also play an important role by binding to the promoter region in pks/nrps gene.What's more,we use GST pull-down technology once again proved that the interaction between LysRLe and Clp protein,and then we made a preliminary study to testify whether Clp and LysRLe protein coordinately regulate HSAF by interacting with each other.We added Clp protein into the EMSA test of LysRLe in the pks/nrps promoter,and the results showed that LysRLe and Clp could enhance the binding of pks/nrps gene promoter to each other.In addition,through the acquisition of ALelysR ?Leclp double mutants of wild-type OH11,we detected the pks/nrps expression of ?Leclp,ALelysR and ?LelysR?Leclp double mutant.Compared with the single mutant ?Leclp and ALelysR,the pks/nrps expression of ?LelysR?Leclp double mutants were further reduced.In conclusion,LysRLu interacts with Clp to regulate the biosynthesis of HSAF.