FUNCTIONAL DOMAINS OF RHIZOBIUM DCT GENES

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The Rhizobium meliloti and Rhizobium leguminosarum dct systems serve as simple models for other bacterial two-component regulation systems. Each has three gene products: a membrane transport protein (DctA); a putative membrane sensor (DctB); and a cytoplasmic transcriptional activator (DctD). DctD has a central domain for activating transcription, an N-terminal domain for inhibiting the central domain, and a C-terminal domain for binding to tandem sites upstream of the dctA promoter. In E. coli, activation of R. leguminosarum dctA but not R. meliloti dctA by DctD requires the integration host factor to bind between upstream bound activator and promoter. The broad goal of this proposal is to learn how rhizobia transform the presence of C4-dicarboxylates into transcriptional activation of dctA, both in free-living and symbiotic states. Specific aims address the following issues. 1) Does DctD bind cooperatively to the dctA UAS, and if so, how? 2) Can DctD bound to either site in the UAS activate transcription, or is this done only by DctD bound to the promoter distal site? 3) Is the central domain of DctD an ATPase? 4) Does the ability to bind ATP distinguish active from inactive DctD? 5) Is phosphorylation of one specific N-terminal domain residue of DctD essential for its activation, or can alternative phosphorylations suffice? These studies involve DNA amplification by PCR, gene cloning, and genetic engineering of the dctA promoter region; over expression and purification of DctD; and biochemical assays for ATP hydrolysis, transcription initiation, and binding of proteins to DNA. Results will contribute to the NIH mission of understanding signal transduction in bacteria, which is relevant to the regulation of virulence properties and many other important bacterial activities. They may also aid improving nitrogen fixation by rhizobia.