The quorum sensing transcriptional regulator TraR has separate binding sites for DNA and the anti-activator

Quorum sensing represents a mechanism by which bacteria control their genetic behaviors via diffusible signals that reflect their population density. TraR, a quorum sensing transcriptional activator in the Rhizobiaceae family, is regulated negatively by the anti-activator TraM via formation of a TraR–TraM heterocomplex. Prior structural analysis suggests that TraM and DNA bind to TraR in distinct sites. Here we combined isothermal titration calorimetry (ITC) and electrophoretic mobility shift assays (EMSA) to investigate roles of TraR residues from Rhizobium sp. NGR234 in binding of both TraM and DNA. We found that K213A mutation of TraRNGR abolished DNA binding, however, did not alter TraM binding. Mutations of TraM-interfacing TraRNGR residues decreased the TraR–TraM interaction, but did not affect the DNA-binding activity of TraRNGR. Thus, our biochemical studies support the independent binding sites on TraR for TraM and DNA. We also found that point mutations in TraRNGR appeared to decrease the TraR–TraM interaction more effectively than those in TraMNGR, consistent with structural observations that individual TraRNGR residues contact with more TraMNGR residues than each TraMNGR residues with TraRNGR residues. Finally, we showed that TraM inhibition on DNA-binding of TraR was driven thermodynamically. We discussed subtle mechanistic differences in TraM anti-activation on TraR activity between homologous systems.

► Quorum sensing transcription factor TraR is inhibited by forming TraR–TraM complex.
► K213 is a key DNA binding residue, but not involved in interaction with TraM.
► Mutations of TraM-interacting TraR residues did not affect DNA-binding of TraR.
► Mutations of TraR residues reduced the TraR–TraM interaction more than those of TraM.
► TraM inhibition on DNA-binding of TraR is driven by thermodynamics.