Identification And Characterization Of Type Ⅵ Secretion System Gene Clusters In Lysobacter Enzymogenes OH11

Lysobacter enzymogenes OH 11 is a gram-negative biocontrol bacterium belonging to Lysobacter genus of Xanthomonadaceae family.OH11,which has no flagella but exhibits twitching motility,separated from the rhizosphere soil of pepper.It not only can secrete chitinase,protease,cellulose and other extracellular hydrolases,but also can synthesize HSAF(Heat Stable Antifungal Factor),a secondary metabolite that can inhibit the growth of various plant pathogenic fungi.It has a strong inhibitory effect on bacteria,fungi and other pathogens.Type VI secretion system(T6SS)is a large injectable molecular weapon widely found in gram-negative bacteria.The structure and mechanism of action in T6SS are similar to the contractile tail of inverted T4 phage,which can inject effector proteins into target cells and secrete homologous immune proteins to gain an advantage in the competition.T6SS usually plays an important role in interspecific competition,toxicity correlation and biofilm formation as an injectable bactericidal weapon.However,the relationship between T6SS and the synthesis of antimicrobial substances in Lysobacter enzymogenes has not reported.With the object of Lysobacter enzymogenes OH11,the research conducted identification and characterization of type VI secretion system gene clusters.We have confirmed that one set of complete type VI secretion system exists conservedly in Lysobacter enzymogenes OH11 by bioinformatics prediction,including 14 core components such as TssJ/L/M membrane complex,TssA/E/F/G/K baseplate complex,Hcp,TssB/C sheath,VgrG,PAARrepeat protein and ATPase ClpV.We found that tssA,tssG and clpV could not be transcribed in LB medium and tssA,tssG,tssM and vgrG3 could not be transcribed in 10%TSB medium through detecting the transcription of T6SS components in LB and 10%TSB medium by RTPCR assay.At the same time,we obtained the mutants of seven important genes in T6SS by gene knockout.The research verified that seven core components of T6SS in Lysobacter enzymogenes OH11 have no affect on the colony morphologies,biosynthesis of yellow pigment,twitching motility and chitinase activity.But the mutant Δhcp not only destroyed the formation of biofilms but also reduced the production of HSAF significantly by studying characterization.The research illustrated that T6SS not only played a positive role in the formation of biofilms but also positively regulated biosynthesis of secondary metabolite in Lysobacter enzymogenes.It provided a new cognitive for the follow-up scientific researchers to study the function of T6SS.We discovered the synthesis ability of HSAF in hcp mutant rescued through genetic complementarity.Hcp had no affect on the growth of strain OH11 by measuring the growth curve,excluding the effect of Hcp on growth kinetics of strain OH11.Importantly,bacterial two-hybrid assay initially illustrated that Hcp interacts with Clp,resulting in regulating the biosynthesis of HSAF indirectly.The result establishes an important foundation for studying the regulatory pathway of Hcp regulating HSAF.