Identification,Interaction And Characterization Of Type ? Pili Assembly And Regulatory Genes In Lysobacter Enzymogenes OH11

Lysobacter enzymogenes belongs to the Lysobacter genus of the Xanthomodaceae family.Differing from other members of the Xanthomodaceae family,Lysobacter doesn't infect animals and plants,it is not the animal and plant pathogenic bacteria,but a type of gram-negative bacteria which is environmentally friendly with potential bio-control.Compared with other members,Lysobacter has another important feature:with no flagella on the cell surface,it has twitching motility by depending on the type IV pili(T4P)(twitching motility,TM).T4P filaments are flexible and mainly composed of structural protein-PilA fimbriae protein(Pilin).T4P are widely distributed in Gram-negative and Gram-positive bacteria,its function involves bacteria toxicity,surface adhesion,motility,biofilm formation,and the genetic material intake,etc.As a typical kind of Lysobacter-Lysobacter enzymogenes had been reported that T4P mediated TM to participate in the bacterium adhesion and colonization of fungal hyphae,it is an important mechanism of bio-control of Lysobacter enzymogenes.Then,there was little knowledge about Lysobacter enzymogenes in T4P assembly and regulation of genes.With the object of independent intellectual property rights to Lysobacter enzymogenes OH11 strains and its complete genome,the research conducted identification and interactions of T4P assembly and regulatory genes.By means of bio informatics prediction,gene knockout,phenotypic characterization and protein-protein interactions,this study identified the main assembly and regulatory genes while forming T4P in Lysobacter enzymogenes,and define the interactions between these gene product,formed T4P assembly model of Lysobacter enzymogenes,these provided an important genetic basis for T4P's biological function(such as hyphae adhesion)in Lysobacter enzymogenes.The main results were as follows:1.The lab found 29 genes associated with T4P in Lysobacter enzymogenes OH11 by bio informatics analysis.They exist in the genome in the form of eight gene clusters.Gene products of gene cluster 1 and 2 are minor pilin which is necessary in forming T4P.They were predicted to be located in intracellular membranes;Gene cluster 3 mainly forms Pil-Chp system,which is responsible for regulation of T4P;Gene cluster 4 mainly forms major pilin(PilA)and the relevant control system(PilS/PilR),etc.;Gene cluster 5 and 8 contain pilT which mediates pilus disassembly;Gene cluster 6 contains a single T4P pilin(PilA2)which is the main major gene;Gene cluster 7 codes the important components(PilMNOPQ)of T4P assembly,whose predicted function is to help PilA assembly in intracellular membranes and periplasmic space.2.We constructed mutant strains lacking 29 pil genes and the corresponding complementary strains by means of gene homologous recombination.Taking these strains as the subject,we adopted various phenotype tests and obtained the following findings:(1)The colony morphology observation showed that significant difference between these mutant strains?pilE1??pilY1??pilX1??pilV1.?fimT??pilG??pilA??pilS??pilR??pilB??pilC??pilM??pilN??pilO??pilP??pilQ??pilT1??pilT2 and wild type strains OH11.Their colonies around moist bright,some strains formed irregular flow state.However,?pilV2??pilW??pilX2??pilY2??pilE2??pilJ??pilL??pilH??pilD?ApilT3 and ?pilA2 theses mutant strains' growth status are similar to wild type strains OH11.(2)The TM detection(the phenomenon of TM:the lolonies' edge have movement cells)displayed that these11 mutant strains ?pilV2??pilW??pilX2??pilY2??pilE2??pilJ??pilL??pilH??pilD??pilT3 and ApilA2 did not lose TM.However,theses 18 mutant strains ?pilE1??pilY1??pilXl1??pilV1??fimT??pilG??pilA?ApilR??pilS??pilB??pilC??pilM??pilN??pilO??pilP??pilQ??pilT1 and ?pilT2 lost TM.That is,no cell movement was found near the colonies,but the corresponding complementary strains can recover TM(clear cell movement can be found near the complementary strain colonies),showed that 18 pil genes involved in the formation of TM in Lysobacter enzymogenes.(3)The scanning electron microscope observation showed that wild type OH11 formed lots of slender T4P on its cell surface.Under the condition of the same electron microscope,we found that 16 mutant strains(?pilEl??pilY1??pilX1??pilV1??fimT??pilB??pilC??pilM??pilN??pilO??pilP??pilQ??pilA??pilR??pilS??pilG)among the 18 mutant strains without TM have no T4P on the surface.This indicated the direct link between T4P and TM.As predicted,we found that ApilTl and ?pilT2 lost TM,but the number of T4P were significantly increased on their surface(compared with WT)as PilT control disassembly of T4P.The results revealed that the T4P on the surface of bacterial cells cannot disassemble to form more T4P when the pilT after missing.Which result that the normal TM mediated by T4P out of order(loss).As controls,3 mutant strains with TM formed clear T4P.(4)The gene transcrption research showed that:the 18 pil genes without TM were in four gene clusters.To be specific:genes from pilEl to fimT in cluster 1 formed an operon and the promoter was located in the upstream offimT.Genes from pilB to pilD in cluster 4 formed and operon and the promoter was located in the upstream of pilB.Genes from pilQ to pilM in cluster 7 formed an operon and the promoter was located in the upstream of pilM.Genes from pilTl to pilT2 in cluster 8 formed an operon and the promoter was located in the upstream of pilT1.3.We investigated interactions of the 18 pil genes by bacteria two-hybird system and the findings were:(1)The core protein PilA of T4P assembly could interact with most Pil protein,which showed that other Pil protein involved in the T4P assembly by PilA;(2)Most products of minor pilins(PilE,PilYl,PilXl,PilV1,and FimT)could interact with each other and they all could interact with the inner membrane pritein PilC of T4P,which indicated PilC could interact with minor pilins in Lysobacter enzymogenes to form the inner membrane platform of T4P and help the core protein PilA's assembly and extension from inner membrane to extracellular;(3)The control gene PilS(a histidine kinase)could interact with PilC and minor pilin.This is a new finding about PilS's perception ofintracellular signal in bacteria and reveals that PilS may require the interaction with PilC or minor pilin to play its regulatory functions;(4)PilT1 and PilT2 are ATPase that mediates pilus disassembly.They can interact with each other but the specific mechanism is unknown.Finally,we put forward the T4P assembly model of Lysobacter enzymogenes by all the Pil protein interactions and other relevant reports.This is the first T4P model in Xanthomonadaceae and lays a foundation for further study of T4P and the pathogenicity of pathogenic bacteria in Xanthomonadaceae and the bio-control mechanism of Lysobacter.