Characteration Of ChpA And PilZ And Mechanism Research Of Their Reguation On Motility In Lysobacter Enzymogenes

Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs T4P(type IV pili)-driven twitching motility to exhibit its antifungal function.Yet,it is still unclear how this bacterium regulates its twitching motility.Here,by using strain OH 11 as the working model organism,we find a hybrid two-component system ChpA and a PilZ domain protein acts as positive regulators in controlling twitching motility in L.enzymogenes.ChpA is a hybrid TCS(two-component transduction system)contains 7 domains including those for auto-phosphorylation and phosphate group transfer,as well as a phosphate receiver(REC)domain.Mutation of chpA completely abolished the wild-type twitching motility,as evidenced by the absence of mobile cells at the margin of the mutant colonies.Further studies of domain deletion and phenotypic characterization reveal that domains responsible for phosphorylation and phophotransfer,but not the REC domain,were indispensable for ChpA in regulating twitching motility.Transcriptomeanalyses of the chpA knockout strain indicated that ChpA was extensively involved in controlling expression of a wide variety of genes(totaling 243).The products of these differentially expressed genes were involved in multiple physiological and biological functions in L.enzymogenes.Thus,we have identified not only a new regulator controlling twitching motility in L.enzymogenes,but also provides the first report demonstrating the broad impact of the conserved ChpA in gene regulation in bacteria.The study found that a variety of PilZ homology domain proteins were involved in bacterial behaviors including twitching motility,pathogenicity,surface adhesion,biofilm formation.a PilZ domain protein were identified in the genome of L.enzymogenes by bioinformatics and genomics.Mutation of pilZ completely abolished the wild-type twitching motility.The alignment of proteins showed that PilZ did not have two N-terminal motifs RxxxR and D/NxSxxG that are involved directly in interactions with c-di-GMP.In vitro,MST assay confirmed that PilZ did not bind c-di-GMP.These interactions between PilZ domain and PilB protein were confirmed in two-hybrid and pull down assays.Mutation of pilB also completely abolished the wild-type twitching motility.Previous studies have shown that PilB is predicted to be an ATPase required for T4P assembly in pil systems.Point mutation assay verify that W71 rather than Y24 is essential amino acid in interaction between PilZ and PilB.E333 and K397 required for PilB enzyme activity are not necessary for interaction with PilZ,but both are essential for PilB involved in twitching motility.In this paper,it provides a theoretical basis that the important role of a hybrid two-component system ChpA involved in twitching motility and its extensive regulation of genes expression to further research on the physiological and biochemical functional mechanisms regulated by the Pil-Chp signaling system in L.enzymogenes.The other regulatory factor PilZ interaction with PilB affect the movement of bacter.further clarification of PilZ combined with PilB regulatory pathways caused by the specific biochemical mechanism.It lay the foundation for further clarification of the PilZ combined with PilB signal pathways caused by the specific biochemical mechanism.