Identification Of Quorum-Sensing System In Mesorhizobium Tianshanense CCBAU 060A And Its Effect On Symbiotic Nodulation

Quorum-sensing signals,mainly referred to N-acyl homoserine lactones,are found in many species of legume-nodulating rhizobia,including Rhizobiurn,Sinorhizobium, Bradyrhizobium,and Azorhizobium,which have been intensively studied.In these genera, quorum-sensing has been linked to various phenomena including nodulation efficiency, growth rate,exopolysaccharide production,and nitrogen fixation,all of which are important for the establishment of a successful symbiosis.In a previous research,we detected AHL signals from an M.tianshanense strain, CCBAU3306,a moderately growing root nodule bacterium.By using a novel method,an AHL synthase gene was obtained from M.tianshanense CCBAU3306 and that quorum-sensing in CCBAU3306 plays a critical role in symbiosis was also proved.In this study,we first investigated AHL production of other tianshanense strains,and found that the AHL profiles of these seven strains were quite different.Most tianshanense strains produced multiple AHL-like molecules and M.tianshanense CCBAU 060A,whose AHL-like molecules contain little small molecules compared with that produced in other tianshanense strains,was selected for further studies.The accumulation of AHL activity in supernatants of CCBAU060A displayed a typical cell density-dependent pattern,with low AHL activity at low cell density and higher activities as the bacterial culture grew.In an effort to identify the genes involved in AHL production in CCBAU 060A,we used transposon mutagenesis to screen for CCBAU 060A strains which detect deficiencies in AHL levels.By screening approximately 5,000 random transposon insertion mutants,six candidate mutants(HJC1,HJC2,HJC3,HJC4,HJC5 and HJC6) were identified as defective in autoinducer production.Cloning and sequencing of the region flanking the transposon insertion showed that the mutant HJC3,HJC4,HJC5 and HJC6 reside in the same regulatory system.One 657-bp ORF encoding a putative protein that is similar to AHL synthase is named mtqI,and another ORF upstream of MtqI encoding a putative protein similar to several LuxR-type transcriptional activators is annotated as mtqR. To study the cell density-dependent expression of the mtqR/mtqI locus,mtqR-lacZ and mtqI-lacZ fusions were constructed.We found that the expression of the mtqI is autoinduced,and none of the mtqI-lacZ fusions are expressed in mtqR or mtqI mutant backgrounds,demonstrating that transcription of mtqI requires both MtqR and MtqI.Another transcriptional regulator mtqS also regulated the AHL production in CCBAU060A.We proved that in the presence or in the absence of additional AHL did not induce mtqI-lacZ expression in mtqS mutant growth.Overexpression of MtqR in the mtqS mutant recovered mostly AHL production in the resulting strain suggests that in CCBAU060A,the expression of mtqR genes is regulated by mtqS,which plays a main role in quorum-sensing regulation.In order to determine whether quorum sensing in CCBAU 060A affects its growth,we compared the growth rate of wild-type strain and the mutant strains.Growth of the mtqR mutant and the mtqI mutant was little delayed compared with wild-type,but the mtqS mutant was even more delayed compared with the mtqR,mtqI mutant.Nodulation experiments suggest that at eighteen days after inoculation,the average number of the nodules per plant in inoculated with the mtqI mutant were less than the wild-type strain,so is the nodulated plants.But sixty four days after inoculation,there is no significantly difference between plants nodulated by the wild-type and the mutant strain.We speculate that quorum sensing plays a critical role in the CCBAU060A symbiotic process.