Studies On Characteristics Of The Quorum-Sensing System In Mesorhizobium Loti And Its Effect On Symbiotic Nodulation

Quorum sensing(QS) is the phenomenon by which bacterial cells can regulate gene expression through monitoring their population density with the detection of diffusible signaling molecules called autoinducers.Rhizobia can infect and invade legumina to form nodules and nitrogen fixation,the process of which is an intricate signal transduction pathway.Recently quorum sensing has been found to play a critical role in the regulation of nodulation and nitrogen-fixing process in a number of rhizobium species,such as nodulation efficiency,biofilm formation,exopolysaccharide production,and nitrogen fixation.As the model stain in Mesorhizobium,the study about the role of bacterial quorum sensing in the nodulation and nitrogen-fixing symbiosis is of a great significance to help us to understand the establishment of micro-plant symbiosis.In the Mesorhizobium loti MAFF303099,analysis of homologous comparison showed that there were at least four annotated acylated homoserine lactone(AHL) synthase genes, which are mlr5638,mlr6103,mlr6385 and mlr9546.In this study,AHL signal molecules could be found in the supernatant of M.loti NZP2213 culture by ultrasensitive bioassay strain JZA1 and displayed a typical cell density-dependent pattern,with low AHL activity at low cell density and higher activity as the bacterial culture grow.The lower AHL activity observed after the highest activity may due to the alkaline hydrolysis in high PH and enzymatic degradation.It indicated that there are quorum sensing symtems in Mesorhizobium loti MAFF303099.The supernatant of M.loti culture was extracted with ethyl acetate and subjected to TLC analysis and electrospray ionization mass spectrometry (ESI MS/MS) analysis,and the results indicated that M.loti produced at least four distinct AHL-like molecules,which were 3-Oxohexanoyl-homoserine lactone(3-O-C6-HSL,MW 214),Octanoyl-HSL(C8-HSL,MW 228 ),decanoyl-HSL(C10-HSL,MW 256) and docecanoyl-HSL(C12:1-HSL,MW 282).Based on the sequence of the M.loti MAFF303099,we designed primers to PCR amplify these 4 synthase genes from the M.loti NZP2213.We successfully amplified three of four putative AHL synthase genes.They were annotated as mrlI1(Mesorhizobium lotiâ… ),mrlI2 and mrlI3,and were homologous to mlr5638,mlr6385 and mlr6103,respectively.In order to study the AHL synthesis of gene mrlI2,3,we expressed the mrlI2 and mrlI3 coding sequences from the P_tac promoter in E.coli using the pYC12 vector,and measured the resulting AHL production.TLC analysis confirmed that while the three AHLs were made by MrlI2,while overexpression of mrlI3 in E.coli was unable to produce detectable amounts of AHLs.To further confirm the role of the mrlI genes,we constructed M.loti strains with in-frame deletions of mrlI2,rnrlI3,mrlI1,2,mrlI1,3,mrlI2,3 and mrlI1-3. Cell-free culture supernatants of these strains were then collected and subjected to TLC assays to examine the AHL content.TLC analysis showed that mrlI2 was responsible for synthesizing the three AHLs,and mrlI3 was responsible for nothing.In order to future study the expression of the autoinducer synthase genes,we constructed two mrlI-lacZ reporters on the broad-host-range vector pRA301,and introduced them into E.coliDH5a and wild type M.loti.The data shows mrlI2 has a little expression and mrlI3 is not expressed.We assume that mrlI2 has a constitute expression and mrlI3 is able to express only being induced in symbiotic process.The physiological assay of the various mrlI mutants showed that quorum sensing system had little effect on the growth and exopolysaccharide formation.Compared with the wide type strain,the numbers of nodules inoculated with the various mrlI mutants were reduced. These data provide strong evidence that quorum sensing plays a critical role in the M.Ioti symbiotic nodulation process,but the exactly mechanism of this is not clear now.