Research Of LuxI/LuxR Type Regulation For Quorum Sensing System In Rhizobium

Quorum-sensing is the phenomenon that the bacteria is able to regulate downstream genes and physical activities through monitoring their own around the cell concentration and the change of other bacteria, with the detection of small diffusible signaling molecules called autoinducers in the surrounding environment. Each QS systems has certain correlation. They have a complex regulatory network, thus it causes the diversity of species., It is currently reported that Quorum-sensing system has been found in many rhizobia. in which QS system of Rhizobium leguminosarum bv. viciae has been studied more detailedly. AS Rhizobium leguminosarum bv. viciae has four QS systems(cin, rai, rhi and tra), thus it made a relatively complex regulatory network. But QS system of Rhizobium etli CFN42 has not studied in detail.This paper mainly uses R. etli CFN42 as the research object. By accessing the MicrobsOnline database, and analysising R. etli CFN42 genomic sequence, we found that there are at least three kinds of AHL autoinduceer biosynthetic genes of luxI type, including cinl, rail and tral. Meanwhile, it includes four kinds of luxR family regulatory genes, including cinR, raiR, traRl and traR1. With the translational fusion vector pRA302, we constructed translational fusion expressing plasmids PluxI-lacZ and PluxR-lacZ, it is found that expression effiencecy of reporter gene lacZ directly reflects the strength of the activated gene promotor. They are then electotransformed into wild-type and luxI/luxR deletion strains. Our study found that cinl expression strictly relies on cinR regulation, rail expression strictly relies on raiR regulation, and tral expression strictly relies on traRl traR2 regulation.In order to get three types of seperated AHLs, we expressed the cinI?rail and tral coding sequence form the Ptac promotor in E. coli (DH5a??pir) using the pYC12 vector. The result is satisfactory that cinI?raiI?tral gene is overexpressed, so we can get many autoinducer AHLs.Through electotransforming PcinI-lacZ, YraI-lacZ and Ptral-lacZ into cinl, rail, tral triple deletion strains, along with adding three kinds of Luxl-type protein producted AHLs into PY medium, then induces expression of reporter gene lacZ. The study found that Cinl-AHLs can induce expression of reporter gene lacZ of all constructed vector in recombinant strains.In order to study the regulatory network, we expressed the cinR?raiR and traR coding sequence form the Ptac promotor in E. coli (DH5??pir) using the pYC12 vector, and electroporated Ptac-cinR,Ptac-raiR and Ptac-traRltraR2 plasmids into cinR, raiR, traR1, tra R2 triple deletion strain, for completing functional coverion and overexpression of cinR, raiR and traR1 traR2. In the meantime, respectively adding three kinds of Luxl-type protein producted AHLs into PY medium with these bacteria, to induce expression of luxI-lacZ. The result showed that Cinl-AHL may combine with all three regulatory proteins CinR, RaiR and TraR1 TraR2, while Rail-AHL only combine with regulatory protein RaiR, and Tral-AHL combine with regulatory protein TraR1TraR2.In words, in the R. etli CFN42 quorum sensing system, cin system is at the top of rai and tra system. cinR is constitutively expression, it may not require any AHLs to induce cinI expression. In case of AHL synthase genes deletion, cinI only expressed in background levels, suggesting that cinI expression may not rely on its own product, and conjecturing that cinR regulates expression of cinI without its own AHLs.In addition, the paper has studied the the effect of sugar to mrh QS system of the slow-growing Mesorhizobium huakuii As9. The exogenous addition of glucose or fructose, could lead to their rapid growth. When exogenous glucose or fructose was added, we found that its expression was significantly lower than that in ordinary culture medium. Therefore, the glucose and fructose could inhibit quorum sensing gene expression in the slow-growing Mesorhizobia huakuii As9. However, with the continuous consumption of carbon source, their inhibition effect towards quorum sensing gene expression will also be reduced.