Molecular Mechanism Of The Regulation Of Mesorhizobium Tianshanese MsiA Expression By MsiR

Mesorhizobium tianshanense is a nitrogen-fixing bacterium which can establish symbiotic assoeiations with Glyeyrrhiza uralensis and form symbiotic nodules on host. In order to establish this mutually beneficial relationship, complicated Signal communication are required. During germination, seed can secret canavanine as an antimicrobial to kill or repress the potential hazard bacteria. M.tianshanense can propagate to be the dominant population paving the way for further symbiosis,cause of MsiAR system.MsiR, a transcriptional regulator for the expression of msiA in response to canavanine, belongs to the LysR-type transcriptional regulator (LTTR) family that is found in diverse prokaryotes. LTTRs regulate a diverse set of genes,including those involved in virulence,metabolisrn,quorum snsensing and motility. Here,MsiR/msiA reagultion system was studied inorder to a better understanding for LTTRs.For studying the regulation mechanism of MsiR on msiA,the recombinant MsiR-his was expressed in Escherichia coli and purified by Tris buffer. By electrophoretic mobility shift assay,we confirmed that the MsiR can bind msiA promoter. The addition of canavanine could enhance the binding strength. At the same time, the interaction between canavanine and MsiR was determined by Isothermal Titration Calorimetry, the stoichiometric number of canavanine with MsiR was 0.86, Kd was 1.86 μM.The transcription start site of msiA gene was determined by 5’race, and then we found the -10 region and -35 region. Five important sites that impacted on MsiR transcription activity was detected by promoter walking and Site directed mutagenesis:RBS-1 (-70,-67),RBS-2 (-59,-56), ABS-1 (-50,-47), ABS-2 (-41,-37).We set a proposed model for MsiR transcripitional regulation.MsiR was tetramer in absence of canavanine and bound to the msiA regulatory region of RBS-1, RBS-2, ABS-2, ABS-3 by a open conformation. At this point, the DNA bending angle is small, MsiR covered msiA promoter -35 region, binding efficiency of RNA polymerase with the promoter was reduced so that the expression of msiA was low. MsiR was tetramer in presence of canavanine and bound to the msiA regulatory region of RBS-1, RBS-2, ABS-2 by a closed conformation. At this time, the DNA bending angle is large, RNA polymerase could bind -35 region, the expression of msiA was high.Finally, We constructed MsiR mutant library, the losing transcriptional activity mutants and higher souble expression at 37 ℃ mutants were screened.