The Roles Of Quorum Sensing Related Genes In Sinorhizobium Sp. 1128 And Their Effects On Symbiotic Nodulation

The previous study of our laboratory has screened an autoinducer synthesis related gene traI in Sinorhizobium sp.1128 by transposon mutagenesis and its coding for amino acid is homologous to(99%identity) the autoinducer synthase traI in Sinorhizobium medicae WSM419.Homologous analysis showed that at least three annotated acylated homoserine lactone(AHL) synthase genes exist in the genome of Sinorhizobium medicae WSM419.So we designed primers to amplify the autoinducer synthase genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences.One of these synthase genes,traI2 was cloned into the expression vector of pYC12 and transformed into Escherichia coli DH5a.It has been detected that at least two different AHLs were produced in the recombinant strain.TLC assay showed that AI production was decressed when traI2 was dirupted.Furthermore,the complementation of traI2 revert the phenotype of AI activity.These findings demonstrate that traI2 is responsible for AI synthesis in Sinorhizobium sp.1128.Homologous analysis also showed that there was a regulatory protein luxR gene up stream of the Smed₁560 gene in Sinorhizobium medicae WSM419.So we designed primers to amplify the luxR gene in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences.By cloned into the expression vector,the LuxR protein was expressed in E.coli BL21.In order to study the regulatory effect between luxR and traI2,we transformed pQY4 (pBBR1-MCS3 derivative carrying intact luxR gene) and pQY6(pRA300 derivative carrying traI2 promoter fragment) plasmids into E.coli MC4100 together by electroporation. Theβ-galactosidase activity assay showed that LuxR protein can intact with traI2 gene in E.coli MC4100 strain,no matter added AI or not.In order to further proved this result we created a traI2-lacZ transcriptional fusion and a traI2-lacZ translational fusion in Sinorhizobium sp.1128,and then over express luxR in these two kinds of mutant.Theβ-galactosidase activity assay also showed the same result. Nodulation result showes that comparing with wild type strain,disruption of autoinducer synthesis related genes,traI and traI2,correlated with an overall decrease in the percents of effective nodules,suggesting a role for quorum sensing in establishing a successful symbiosis between the bacterium Sinorhizobium sp.l128 and its plant host Melilotus suaveolens Ledeb.