Mechanisms Of Two-component System PilS-PilR In Regulating The Biosynthesis Of Antibiotic HSAF And Type ? Pili Formation In Lysobacter Enzymogenes OH11

The genus Lysobacter is an aerobic Gram-negative bacterium belongs to y-proteobacteria,Xanthomonadaceae family.This genus of Lysobacter is named after its unique lytic activity,as it produces abundant extracellular enzymes to degrade the cell walls of pathogens such as bacteria,fungi,nematodes,oomycetes and algae.In addition,Lysobacter exhibits broad-spectrum antimicrobial activity through synthesis of various secondary metabolites.It makes the bacterium to function not only as a promising source of novel bioactive natural products,but also as an effective biocontrol agent in the industrial and agronomic applications.Previous studies showed that the biocontrol functions of Lysobacter are affected by nutrition conditions in the survival environment.It possibly adopts a certain way to perceive environmental nutrition signals during the interaction processes with pathogens,and inhibits the growth of pathogens through producing a variety biocontrol factors.Two-component system is the most prevalent method to sense and respond to intra-or extracellular stimulus in bacteria.The canonical two-component system is composed of histidine kinase and response regulator.Upon receipt of signal stimulus,the histidine kinase catalyzes an autophosphorylation reaction and then transfers the phosphoryl group to its cognate response regulator.The phosphorylation of response regulator causes protein conformation changes and activates the output domain to elicit specific regulatory functions.Therefore,Lysobacter possibly adopts various two-component systems to sense the changes of environmental nutrition,and then adjusts its biocontrol functions.The knowledge about the biocontrol traits and regulatory mechanisms of Lysobacter is still poor since found in 1978,compared with the other biocontrol bacteria such as Bacillus and Pseudomonas.Among the 45 species of Lysobacter found so far,the most well studied strain is L.enzymogenes.It exerts antagonism against pathogenic fungi via two biocontrol factors.First,this strain utilizes the synthesized sencondary metabolite,named as heat-stable antifungal factor(HSAF),to exhibit well antagonistic activity against pathogenic fungi.The other is twitching motility mediated by type IV pili,which is beneficial for L.enzymogenes to colonize and adhere to fungal hyphae.It will promote the infection of fungal hyphae by L.enzymogenes and finally inhibit the growth of fungi.In this study,we used bioinformatic method to analyze the genome of L.enzymogenes strain OH11.Finally we identified 48 histidine kinases and 53 response regulators.Then we deleted the genes encoding response regulators individually and constructed a genome-wide deletion mutants library in L.enzymogenes.Through analyzing HSAF yields and twitching motility of each deletion mutant in the library,we determined the response regulators which make important contribution to the biocontrol functions of L.enzymogenes.The results revealed that PilR,AlgR and FimX are essential response regulators for twitching motility formation in L.enzymogenes.All of the three proteins are conserved regulatory proteins responsible for type IV pili formation in bacteria.Also,deletion of pilR and cheB significantly decreased the production of HSAF,which indicated that both two genes positively regulate HSAF biosynthesis in L enzymogenes.This is the first report about PilR and CheB regulating the biosynthesis of secondary metabolites in bacteria.Through systematic analyses of the functions of response regulators in L.enzymogenes,we recognized that the response regulator PilR simultaneously regulates HSAF biosynthesis and twitching motility.Deletion of the upstream gene pilS also shows the same phenotype,indicating that PilS-PilR constitute a pair of typical two-component system and play an important role in the biocontrol function of L.enzymogenes.For this reason,we further explored the mechanisms of PilR in regulating the two biocontrol factors and found that PilR independently regulates HSAF biosynthetic and type IV pili formation pathways.When regulating type IV pili formation,PilR directly binds to the promoter region of pilA,which is the key gene for type IV pili biosynthesis to control the transcription level,and finally effects twitching motility of L.enzymogenes.While,PilR regulates HSAF biosynthesis via indirectly effecting the intracellular concentration of the second messenger c-di-GMP.For the first time,this study has explored the effects of two-component systems on biocontrol factors produced by the biocontrol bacterium L.enzymogenes at macro-and microscopic levels.We systematically identified and screened the systems which exert essential function on biocontrol factors.Besides,we made an in-depth exploration about the regulation mechanisms of PilS-PilR two-component system.Finally,we revealed a novel regulating pathway of HSAF biosynthesis which is independent of type ? pili formation pathway.The results will provide theoretical guidance for the genetic improvement of L.enzymogenes using two-component systems as targets.On the other hand,it provides experimental evidences and theoretical bases to enrich the knowledge about the functions and regulation mechanisms of two-component systems in bacteria.