Identification And Functional Characterization Of A Toxic Effector Protein Le1519 Of The Type Ⅳ Secretion System In Lysobacter Ennzymogenes

Lysobacter enzymogenes OH11 is a promising agricultural biocontrol bacterium isolated from pepper rhizosphere soil.L enzymogenes OH11 secretes an antifungal secondary metabolite,known as the heat-stable antifungal factor（HSAF）to fight against numerous fungal pathogens.Via HSAF,L.enzymogenes is effective in controlling several fungal disease of crop plants in field,including fruit tree canker and wheat scab.However,L.enzymogenes OH11 fails to inhibit the grwoth of plant pathogenic bacteria by secreting antibaterial secondary metabolites.Previous studies from our laboratory found that L.enzymogenes OH11 could efficiently kill multiple bacteria through a cell-cell contacting-dependent machine,known as the type IV secretion system（T4SS）.In pathogenic bacteria,the contat-dependent T4SS is known to kill competitor bacteria by means of translocating toxic/anti-bacterial effector proteins.Thus,the objective of this study is aimed to identify T4SS toxic effector proteins from L.enzymogenes OH11,a biocontrol agent.Smlt3024 from the animal pathogenic bacterium,Stenotrophomonas maltophilia is an experimentally-validated,RTX-like toxic effector translocated by the S.maltophilia T4SS.Sequence alignment revealed that the genome of L.enzymogenes OH11 carried a Smlt3024homolog,Le1519 that shared 43%amino-acid similarity with Smlt3024.Detailed sequence analyses showed that like Smlt3024,Le1519 also possessed a conserved C-terminal domain termed as the XVIPCD that is a distinguished domain of effector proteins transolicated by the contact-dependent killing T4SSs.To understand whether Le1519 may induce toxicity to competitor bacteria,this study established an arabinose-induced gene-expression system in the model bacterium,Escherichia coli（E.coli）.We found that the arabinose-induced expression of Le1519 in the periplasm of E.coli had no toxic effect,as this step did not inhibit the growth of E.coli.However,the arabinose-induced expression of Le1519 in the cytoplasm of E.coli remarkably inhibit the grwoth of E.coli and this growth inhibition is independent of the XVIPCD domain carried by Le1519,suggesting Le1519 is possibly tranlocated by L.enzymogenes into the cytoplasm of competitor cells,i.e.E.coli.This finding is different to the mode of action of Smlt3024,as earlier study documented that Smlt3024 functions in the periplasm of competitor cells.Previous studies identified two critical characteristics of effector proteins translocated by the contact-dependent killing T4SSs:（i）T4SS effectors employ their XVIPCD domain to physically interact with Vir D4,a T4SS-pathway specific ATPase.Such protein-protein interactions supply enery to enable T4SS effector transcolation by the ATP hydrolysis via Vir D4;（ii）toxicity of T4SS effectors could be neutralized by neighbor immunity proteins through direct protein-protein interaction manners.Via bacterial two-hybrid and pull-down approaches,we found that Le1519 indeed interacted with Vir D4 and this protein-protein binding is dependent on the presence of the XVIPCD domain of Le1519.We also found that Le1518 encoding a hypothetic protein is the immunity protein of Le1519,as co-expression of Le1518 and Le1519 neutralized the toxicity of Le1519 in the cytoplasm of E.coli and both proteins indeed occurs direct binding.Taken together,above findings popssible rcognzied by the respective immunity protein of Le1519 in L.enzymogenes.To further validate above findings,the Le1519-GFP and Le1518-m Cherry fusion proteins were constructed,respectively.Fluorescence observations showed that Le1519-GFP mainly located at the pole of the E.coli cell,whereas the Le1518-m Cherry was found to be distributed in the whole cell of E.coli.Co-expresion with Le1519,localization of Le1518 in the E.coli cells shifted from whole cell to pole,suggesting Le1519 possibly recruits Le1518,by which Le1518 forms a protein complex wih Le1519 to neutralize the toxicity of Le1519 in E.coli.Moreover,we also identified a non-toxic Le1519 variant(Le1519^T129A)that represents the native Le1519 occurs an amino residue（threonine,T）substitution at position of 129 by alanine（A）.While the arabinose-induced expression of Le1519^T129Ain the cytoplasm of E.coli had no toxic effect,however,Le1519^T129Ais similar with the native Le1519 in polar localization and the binding with the immunity protein Le1518,revealing the toxicity of Le1519 is possibly independent on its polar localization.Finally,to understand how Le1519 induced cell death in E.coli,we aimed to identify what E.coli components mediate the toxicity of Le1519.To achieve this,an E.coli mutant library was generated by using the mini-Tn5 transposon mutation technology.We screened the library and aimed to select the E.coli mutants that carried Le1519 but survived in the presence of arabinose.This step led us to identify such a mutant which had a mutation in the E.coli ira M gene.Ira M encodes an anti-adapter protein,whose function is to inhibit the Rpo S proteolysis by regulating Rss B activity,thereby increasing the stability of the sigma stress factor Rpo S（σ^s）during magnesium starvation.To confirm this finding,we generated an in-frame deletion mutant of ira M at the wild-type background of the E.coli Top10 strain.At this mutant,the toxicity of Le1519 was found to be significantly decreased compared to the wild-type E.coli Top10 strain,suggesting Ira M indeed participated in the toxic signaling of Le1519 in E.coli.In conclusion,this study,for the first time,identified a T4SS toxic effector protein,Le1519 from the biocontrol L.enzymogenes OH11.Le1519 physically interacted with Vir D4,by which Le1519 is potential recognized by the L.enzymogenes T4SS.Le1519 also bound and formed a protein complex with its immunity protein Le1518 by which its toxic effect to its native host is neutralized.Finally,the E.coli Ira M was identified to be invoved in the toxic signaling of Le1519 in E.coli.These results collectively suggest that L.enzymogenes could utilize the contact-dependent killing T4SS to kill competitor bacteria via injecting toxic effectors（i.e.Le1519）,which represents a novel biocontrol mechanism of L.enzymogenes independent of the well-recognized secondary metabolites.