Functional Analysis Of Quorum Sensing Regulator MrtR In Mesorhizobium Tianshanense And MrlI1-MrlR1 Quorum Sensing System In Mesorhizobium Loti

As one of the most important signal transduction pathways in bacteria, quorum sensing system is involved in many regulatory circuits in rhizobia, especially the communication between rhizobia and their plant hosts. The typical QS system in Gram-negative bacteria is the marine symbiotic bacteria Vibrio fisheri LuxI/R-type regulatory system. The key regulatory components of these signaling systems are LuxI-type AHL synthases, and LuxR-type AHL receptors and AHL-dependent transcription factors. Here, both of the regulatory mechanism of quorum sensing regulator MrtR of Mesorhizobium tianshanense CCBAU 3306 and Mrlll/MrlRl quorum sensing system of Mesorhizbium loti NZP 2213 were studied.Mesorhizobium tianshanense, a nitrogen-fixing symbiont for at least eight different plant species, including Glycyrrhiza (licorice), produces most diversity of AHL signaling molecules. Interestingly, the MrtI/R quorum sensing system in M. tianshanense seems to be responsible for the synthesis of all of the AHL molecules and controls its symbiotic nodulation. To further study the role of MrtI/MrtR QS system during the symbiosis process of M. tianshanense and Glycyrrhiza, the structure and function of MrtR was analyzed in detailed. Detected by the electrospray ionization mass spectrometry (ESI MS/MS), the acyl homoserine lactone (AHL) signal molecules produced by M. tianshanense (MAI) were suspected to be 3-oxodocecanoyl-HSL (3-O-C12-HSL, MW300) and 3-oxotetradecenoyl-HSL (3-O-C14-HSL,MW 326). And MrtR can specifically bind with the mrt box inside the promoter region of mrtI in vitro and activate the expression of mrtI in vivo by binding with the autoinducer,3-O-C12-HSL (MAI).The way of mrtI expression regulated by MrtR shown that mrtI could only be expressed when the predicted mrt box, the invert repeated sequence located in the-69 to-52 region of mrtI promoter, was bound by MrtR-MAI complex. That is MrtR, MAI and mrt box were indispensable for expression of mrtI, which is also the typical characteristics of regulatory mechanism of LuxR-type protein.To determine the regions of MrtR necessary for transcriptional activation of mrtI, a serial of deletion mutations of MrtR were generated. Eight MrtR mutant encoding truncated MrtR proteins with deletions of residues of 2-30,2-40,2-50,2-80,2-120,2-140,2-160 and 230-272 were constructed by PCR amplying and cloning into pBBR-MCS5 vector under the control of Plac promoter. All of these constructs were transformed into a M. tianshanense MrtR/MrtI quorum sensing system deleted mutant MHT9 strain, with PmrtI-lacZ (pMH1) report gene. Based on analysis of the activity of LacZ, the result showed that similar to C-terminal truncations of LuxR, RhlR, and TraR, MrtRΔ233-271 which lacks the conserved HTH DNA binding domain, had no activity. And MrtRΔ2-30 was similar to that of full-length MrtR. However, deletion of up to 50 N-terminal amino acids enhanced MrtR activity even in the absence of 3OC12-HSL. MrtRΔ2-80,Δ2-120, andΔ2-140 showed no activity; however, MrtRΔ2-160 displayed a low level of autoinducer-independent activity. Previous studies of LuxR and TraR suggest that in activators of the LuxR-family, the N-terminal domain masks the C-terminal DNA binding domain in the absence of autoinducer, thus interfering with DNA binding, and the C-terminal domain alone was shown to function in AHL-independent activation.To further characterize the structure and function of MrtR, we screened for MrtR constitutive mutants. We used E. coli mutator strain XL-1 Red to introduce mutations to the Plac-mrtR plasmid. The mutated plasmids were then transformed into M. tianshanense MHT9 strain containing a PmrtI-lacZ plasmid, and the transformants were spread on X-gal plates without any autoinducer. Approximately 25,000 colonies were screened, of which exhibited a LacZ+ phenotype in the absence of autoinducer and had point mutations in the mrtR gene. Compared to wild-type MrtR, all eleven MrtR mutants had enhanced ability to activate the expression of mrtI in the absence of 3OC12-HSL. However, a cluster of mutations located in the C-terminal DNA binding domain (N204D, P208S, H219R, T221M, and Y227C) were hypersensitive mutants as they still responded to the 3OC12-HSL stimulation. The constitutive MrtR mutants that activated mrtI independent of and with no further stimulation by 3OC12-HSL were clustered in the N-terminal autoinducer binding domain (S6L, V105A, G114S, M135I, H142R, and R182C). We further characterized the mutant, MrtRG114S, with the highest activity in the absence of 3OC12-HSL by purifying it as His6-tagged recombinant protein. The MrtRG114S protein could efficiently retard mrtI promoter DNA in the absence of 3OC12-HSL.The second part of this desertation is about the study of MrlRl/Mrlll quorum sensing system of Mesorhizobium loti NZP2213. Detected by ultrasensitive bioassay strain JZA1, AHLs could be found in the supernatant of M. loti NZP2213 culture in TY medium (not, in MM medium) and displayed a typical cell density-dependent pattern, which indicated that there was quorum sensing system in M. loti NZP2213. The supernatant of M.loti culture was extracted with ethyl acetate and subjected to TLC and electrospray ionization mass spectrometry (ESI MS/MS) analysis, and the results showed that M. loti could produce at least four distinct AHL-like molecules, which were suspected to be3-Oxohexanoyl-homoserine lactone (3-O-C6-HSL, MW 214), Octanoyl-HSL (C8-HSL, MW 228), decanoyl-HSL(C10-HSL, MW 256) and docecanoyl-HSL (C12-HSL MW 282)。Based on the sequence of mlr5638 and mlr5637 of M. loti MAFF303099 strain, autoinducer synthase gene mrlI1 and relevant transcriptional regulator mrlR1 from the M. loti NZP2213 were successfully amplified by PCR. mrlI1 and mrlR1 were 95% and 92% homologus to mlr5638 and mlr5637 at amino acid level respectively. By hetro-expressing mrlI1 in E. coli and constructing a mrlll deletion mutant in M. loti genetic background, we found that MrlI1 was responsible for synthesizing AHL molecules with long carbon train (C12-HSL) in M. loti and the expression of mrlI1 was shown to be growth phase-dependent, regulated by MrlR1 and the autoinducer produced by the protein itself, which is of typical autoinducing and quorum sensing charateristics. To examine whether the MrlR1/MrlI1 quorum sensing system would affect nodulation, nodulation assays of AHL-deficient mutants on their native plant host Lotus japonicus was performed and it was found that the efficiency of nodulation was reduced in mutation of mrlI1, suggesting that quorum sensing system in M.loti may play an important role in successful establishment of a rhizobium-legume symbiosis.