Research For The Effect Of Cin Quorum Sensing System On Symbiosis And Exopolysaccharides Systhesis In Rhizobium Etli CFN42

Many bacteria are able to produce signal molecules in reponse to cell density,which is referred as autoinducers.They can induce the expression of the downstream genes,while a critical concentration has been reached.The progress is often termed as quorum sensing.In R.etli CFN42,cin system is at the top of rai and tra system.QS regulates a variety of physiological functions,which involving in the production of exopolysaccharide and symbiosis.EPS is essential to infect the legumes root.The systhesis of EPS in Rhizobium leguminosarum and Sinorhizobium meliloti is closely associated with QS system,which furthermore influences the nitrogen fixation of nodules.This paper used R.etli CFN42 as the research object,and the finding,that cin system is at the top of the QS regulatory network,as the basis.The first part of the paper focused on the influence of cin system on the symbiotic process in R.etli CFN42.First of all,the colonization of WT and cinR mutant in the vermiculite,lawn soil and clover was studied,which indicating that the mutant in cinR doesn't impact the colonization of R.etli in soil.We then inquired into symbiotic nitrogen fixation capacity of WT and cinR/I mutants in the vermiculite.It turned out that the mutant in cinR can impair the ability of nodulation and nitrogen fixation.In the experiments of symbiotic nodulation in the lawn soil without rhizobia,it was found that there is no difference in the nodule number formed by WT and cinR mutant at the inoculation concentration of 104/g soil,while the cinR mutant formed less nodules than WT at the concentration of 102/g soil.Furthermore,the nodules formed in the natural soil were more than which were formed in the sterile soil,which implied that the native bacteria in the soil may help with R.etli in nodulation.Ultimately,the competitive nodulation of WT and cinR mutant in the clover soil with rhizobia was researched.The results showed that the phenomenon of nodulation is similar with that in the lawn soil.In the competitive nodulation experiments,cinR mutant showed lower nodule occupancy than WT,indicating that cinR mutant owns defect in competitive nodulation.The nodule occupancy of WT and cinR mutant increased at high inoculation concentration,implying that the high population density of rhizobia plays key roles in the process of competitive nodulation.The second part of the paper focused on the influence of cin system on the EPS production in R.etli CFN42.When culturing on the YEM medium,the colonies formed by cinR/I mutant is smaller than WT.Followed by ethanol sedimentary method to extract the EPS generated by R.etli CFN42,the anthrone-sulfuric acid method was used to quantify the EPS.The result indicated that there is no significant difference in EPS production between WT and cinR mutant.When growing on the Ml medium for 4 d,the mutation in cinR and cinl cause the emergence of the "droplet" phenotype,while WT doesn't appear this phenotype.However,after continuing growing for 4 d,the "droplet" phenotype wears off.It turned out that cin system has little effect on the output of EPS,but mainly affect the construction of EPS.In order to screen the downstream genes controlling this phenotype and regulated by cin system,we used the method of the conjugation between WT or cinR mutant and SM10?pir(pSC123),to inactivate some gene by transposon insertion.In the WT background,there were three genes inserted by transposon more than once-cinR,hipB and COG5001,indicating that the screening result is credible.Followed by the contruction of the translational fusion plasmid-hipB::lacZ and COG5001::lacZ,the expression level of hipB and COG5001 were detected in WT and cinR mutant.It indicated that the expression of hipB is repressed by cinR,while cinR doesn't have effects on the expression of COG5001.Moreover,the phenotype of biofilm enhancement emerged in the case of the mutation in hipB.It was speculated that the hipB plays a similar role in R.etli CFN42 compared to the praR in Rhizobium leguminosarum bv.Viciae and CinR could relieve the repressive effect of HipB on the expression of the downstream genes.