Function of MsiR on canavanine-mediated repression in Mesorhizobium tianshanense

Mesorhizobium tianshanense employs MsiA as canavanine exporter, which is upregulated by MsiR, to successfully form a symbiosis with the legume Glycyrrhiza uralensis. In this research, through gel-shift and bacterial two-hybrid examination, MsiR was found to spontaneously form dimers and bind to msiA promoter without additional canavanine. Six truncated forms of MsiR were constructed, and the conserved helix-turn-helix (HTH), substrate-binding, and surface-loop domains were found essential for MsiR functions. Random mutagenesis was used to study the functional sites of MsiR. Seven point mutants were selected, in which three mutants constitutively induced msiA expression without additional canavanine, two mutants partially changed substrate specificity, and the other two were almost null mutants. Results from the site mutation show that the functional subunits (HTH domain, dimerization interface domains, and C-terminal) are important in the conformation and induction ability of MsiR.