Effect Of LuxR/LuxI Type Regulator In Rhizobiumetli On AHLs Activities, Exopolysarides Production And Symbiosis

R.etli forms nitrogen-fixing nodules on the roots of of common bean, quorum sensing in R. etli is fragmentary but seems as complex as that of R. leguminosarum bv.viciae which had been best characterized. Till now, only tra genes involved in self-conjugative plasmid transfer was detailed described in R.etli CFN42. In another R.etli strain, CNPAF512, raiRI and cinRI had been reported, but the structure of these AHLs moleculses is still unkonwn but that cinRI for Hydroxylated long chain, raiRI for Short chain AHLs synthesis. In this work we identified the structures of all AHLs produced by three different synthase respectively, and show that these quorum sensing gens are involved in exopolysacchrides prodtion and symbiosis interation.Search for the genome sequence of R. etli CFN42 was performed, found other two genetic LuxR/I homologues indenpdent with Tra, designated CinR/I and RaiR/I, respective-ly locate on R.elti choromose and plasmid p42f. A serises of single,double, triple luxI/luxR in-frame deletion were constructed by overlapping PCR of flanking regions and cloned into the sacB suicide vector pEx18Gm to select for double-crossover events.To further study the roles of QS system of R. etli, the structure of AHLs molecules produced three LuxI synthases were analyzed in detail. The electrospray ionization mass spectrometry (ESI MS/MS) anyalisis conbined by thin-layer-chromography revealed that, Four AHL molecules were synthased by R.etli, Rail produced 3-OH-C8-HSL, Tral produced two components C8-HSL and3-oxo-C8-HSL, while CinI synthased long-chain AHLs:3-OH-C10-HSL. To investigate the functions of the three LuxI/R regulartory gene, we examined AHL prodution in a series of single, double, and triple mutants with in-frame deletions of QS genes. Mutation of single cinl or cinR surprisingly resulted in dramatic decrease of AHL accumulation, remaining about 1/100 AHL activity of that produced by wild type strain at early-stationary phase, this indicate that cinl and cinR may suite at the top of the QS system to regulate rai and tra QS genes.Mutations of rail and raiR did not significantly alter the AHLs activities observed in the parent stain. The loss of tral greatly reduced the production of putative AHLs detected, even changed the pattern of AHL production curve. Theses resutls imply that in R.etli, enzyme activity of Tral/R may be higher than RaiI/R, or for the AHLs detector, Agrobacterium tumefaciens, which has a bias for 3-oxo-C8-HSL which produced by TraI homologue.Plac-expressed luxl and luxR genes were introduced into responding QS mutants to determine AHLs activities produced by these complementary strains. All luxI or luxR type gene except cinR could restore AHLs production from luxI/luxR single mutant. CinI produced very lower AHLs activity in luxl triple mutant indicate that CinI synthase may was a lower-expressed enzyme in R.etli.In a QS system, active LuxR often act as a regulator by binding DNA region of specific target genes. The LuxR mutant was further investigated for phenotype defect caused by QS. Mutation of cinR or cinl gene lead to decreased EPS prodution and weaker biofilm formation in R.etli.The effect caused by cin QS system may mediated by transcriptional repressor RosR which could regulate genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism.Symbosis phenotype of luxI/luxR single and triple mutants were investigated and revaled that no significane difference was observed in all QS mutans in first nodule apperance. cinR mutant showed significiant less noduled number but formed much more pink and big nodules than other treatments.RaiR formed less number of big pink nodules. cinR,raiR,traI also showed lower nitrogen fixting activities. The observation indicated that QS may play some roles in rhizobia-plant interation.We further investigated the competitiveness of three luxR mutant. All co-inoculation with WT and luxR could form more nodules and lead to increased dry nodule weight. When coinoculated with WT at a ratio of 1:1 in cell density of 108/ml, all luxR mutants showed high competitiveness. When inoculated with WT iat a ratio of 0.1, cinR showd same nodule capacity with WT, but in ratio of 0.01, cinR will lose infection ability. These results indicate that the strong competive ability of cinR could be decreased by cell number disadvantage. Above all, loss of LuxR, especially CinR, may have a advantage for nodulation in R.etli, to convenient host infection or other physical behavior.