Study Of The Interaction Between Msir And Dna Or Co-inducer

Mesorhizobium tianshanense is a nirogen-fixing bacterium which can establish symbiotic associations with Glycyrrhiza uralensis licorice.During germination of licrice,seed can release canavanine as an antimetabolite to eliminate the potential pathogenic microorganisms in soil.While,M.tianshanense can survive and thrive in the hostile environment due to the msiA/msiR system which can export the canavanine.LysR-type transcription regulator(LTTR)MsiR in M.is required to active transcription of the msiA gene in response to canavanine released by host plant licorice.We put more attention to the interaction between the MsiR and DNA or canavanine in this study,the research may provide inspiration to uncovering regulatory mechanism of LTTRs.During protein homology modeling and docking,six amino acid residues(N102?D104?D131?D202?F230?M242)which closely related to binding canavanine were predicted.All mutants showed lower affinity for canavanine by ITC assay,M242A mutant decreased the affinity by about 1 time,and the titration process was a exothermic process and enthalpy favorable,while the affinity of D131 A?D104A?D202A?F230A mutants were respectively decreased by 4.6 times?22.9 times?30 times and 39.3 times,and the titration process were all exothermic processes and entropy favorable,the activity of the M242A mutant decreased about 27%and the other five mutants almost lost the ability to transcriptionally activate msiA expression.Our data suggests that the six amino acid residues were critical for MsiR binding canavanine.We then mutated each polar amino acide residues,which may play a role in the DNA binding,to alanine on a3 of MsiR,respectively.The transcriptional activity of mutants in this part were all decreased in different extent except the N27A mutant.We also successfully obtained seven MsiR mutants which could active transcription of msiA in the absence of canavanine through random mutagenesis and double screening system.Through random mutagenesis at ABS1 site of msiA promoter region,we found some constitutive promoters with an ACAN consensus sequence,and then identified a new promoter composed with ACAN and ABS3 site by 5' RACE assay.We also found ATTA,GTTA and ATAA mutated promoters at ABS1 site which could efficiently respond to canavanine as wildtype;GATA,GTTC and CTGA mutated promoters only had half transcriptional activity of wildtype under the same canavanine concentration.Five of the promoters which lost transcriptional activity were found to bear at least three bases different from wildtype or high inducible promoters by sequencing.Our data suggests that the ABS1 site may play an important role in the process of MsiR activating transcription of msiA gene in response to canavanine.