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 hetero-complex. 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 TraR(NGR) abolished DNA binding, however, did not alter TraM binding. Mutations of TraM-interfacing TraR(NGR) residues decreased the TraR-TraM interaction, but did not affect the DNA-binding activity of TraR(NGR). Thus, our biochemical studies support the independent binding sites on TraR for TraM and DNA. We also found that point mutations in TraR(NGR) appeared to decrease the TraR-TraM interaction more effectively than those in TraM(NGR), consistent with structural observations that individual TraR(NGR) residues contact with more TraM(NGR) residues than each TraM(NGR) residues with TraR(NGR) 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. (C) 2012 Elsevier Inc. All rights reserved.