Tandem DctD-binding sites of the Rhizobium meliloti dctA upstream activating sequence are essential for optimal function despite a 50- to 100-fold difference in affinity for DctD.

The Rhizobium meliloti genes dctB and dctD positively regulate the expression of dctA, which encodes a C4-dicarboxylate transport protein. Here we characterize an element (UAS) located upstream of dctA that has tandem binding sites for the dctD gene product (DctD). At relatively low concentrations of active DctD, the element activated dctA transcription, but at relatively high concentrations of DctD it was inhibitory. The UAS failed to function when placed further upstream of dctA. Both DctD-binding sites were required for optimal UAS function, despite a 50- to 100-fold difference in binding affinities. Moving the promoter distal binding site 5 bp further upstream was functionally equivalent to its deletion. Based on these data, we hypothesize that the sigma 54-dependent activator DctD binds co-operatively to the R. meliloti dctA UAS, and that occupancy of both sites is required for maximal activation of dctA.