Identification And Function Characterization Of Quorum Sensing System In Lysobacter Enzymogenes

Lysobacter enzymogenes strain OH11, isolated from cayenne-root soil, is reported forthe first time as a bacterial biological control agent. It is characterizaed with a highG+C content and gliding motility. Lysobacter enzymogenes produce extracellular lyticenzymes, in including chitinase, protease, cellulase and β-1,3-glucanas and some kind of.So, it displays in vitro activity against fungal and oomycetous plant pathogens. The objectives of this work are to:(i) whether the DSF (diffusible signal factor) style QS (quorum sensing) exist in Lysobacter enzymogenes;(ii) how the DSF make its role in Lysobacter enzymogenes;(iii) make clear the difference between the L. enzymogenes and other Xanthomonas spp. on the function of rpfF.With the genomic information of Xanthomonas in NCBI database, we found the rpf gene cluster in Lysobacter enzymogenes for first time though homology comparimarison.The gene cluster is in charge of DSF biosynthesisand transduction.The result shows that Lysobacter enzymogenes share the similar functional domain with other Xanthomonas. So,we Speculated that the function of rpf gene cluster may be similar to Xanthomonas spp. In order to verify the result, we knockouted the rpfF and rpfG gene from the rpf gene cluster.In Xanthomonas spp., the function of DSF style QS system is very comprehensive, It can control the pathogen in the host pathogenicity, pathogenic factors (such as extracellular enzymes and extracellular polysaccharides), biofilm formation, migration and other important characters. Lysobacter enzymogenes belongs to the family of Xanthomonadacae just like Xanthomonas spp., but it is identified as a biocontrol bacteria with no pathogenicity. In the rpfF gene is in charge of molecule synthesis of DSF. We transfered the rpfFLys into Xcc rpfF mutant by genetic operation,The ability of protease and DSF decrsed for rpfF missing,but the rpfFLys could restore the drop.To make confirmation of the result, we knockouted the rpfF gene through homologous recombination. Comparied with the wild type, the rpfF mutant stain reduced the ability HSAF production and antagonistic activity against Pythium. The rpfF mutant stain reduced the production of WAP and the ability against Clavibacter michiganense The result of high performance liquid chromatography and fluorescence quantitative PCR coincided with and HSAF activity detection and Pythium antagonist.In addition, we test the gene expression difference between wild type and rpfF mutants though microarray. The results showed that the expression of384genes were influced for rpfF missing. However, the mutation doesn’t change enzyme activities of four exocellular antibacterial hydrolase of Lysobacter enzymogenes. Moreover, we find something interesting that although mutation may lead to acrid surface of colonies, its sliding ability is strenghtened.It has been proved that rpfC/rpfG constitute a two-component regulatory system which is used by the Xanthomonas spp. to percept and transducte the quorum-sensing signal molecule (DSF). In the charge of intracellular delivery of DSF, rpfG regulates pathogenicity of Xanthomonas spp.. In this study we focus on the analysis of function of rpfG in Lysobacter enzymogenes. Set OH11as research object, we identified its paralogous gene of rpfG through sequence homology analysis and named as rpfGLys. With homologous recombination technology, we get the depletion mutant of rpfG by carefully directed knockouts. Compared with the wild type, the mutant has a low secretion of heat stable antibacterial factor (HSAF) which is an important antibacterial substance of Lysobacter enzymogenes and a lower antipathogenic activity toward Pythium aphanidermatum. Fluorogenic quantitative PCR shows that expression of the key gene (PKS/NRPS) which is in charge of biosynthesis of HSAF is significantly lower, which coincide with activity detection of HSAF. Further researches find that the mutation doesn’t change enzyme activities of four exocellular antibacterial hydrolase of Lysobacter enzymogenes. Moreover, we find something interesting that although mutation may lead to acrid surface of colonies, its sliding ability is strenghtened.