FUNCTIONAL DOMAINS OF RHIZOBIUM DCT GENES

苜蓿根瘤菌和豆科根瘤菌dct系统用作 其他细菌双组分调节系统的简单模型。 每个 有三种基因产物：膜转运蛋白（DctA）; 膜传感器（DctB）;和细胞质转录激活因子（DctD）。 DctD具有用于激活转录的中心结构域、N-末端结构域和N-末端结构域。 用于抑制中心结构域的C-末端结构域，和 与dctA启动子上游的串联位点结合。 在大肠大肠杆菌， 激活R.豆科根瘤菌DctA而非根瘤菌R.苜蓿dctA通过DctD 需要整合宿主因子与上游因子之间的结合 激活剂和促进剂。 本提案的总体目标是学习如何 根瘤菌将C4-二羧酸的存在转化为转录 dctA的激活，无论是在自由生活和共生状态。 具体 目标涉及以下问题。 1)DctD是否与 dctA无人机系统，如果是，怎么做？ 2)DctD是否可以绑定到UAS中的任一站点 激活转录，还是仅通过与启动子结合的DctD完成 远端部位？ 3)DctD的中心结构域是ATP酶吗？ 4)是否 结合ATP的能力区分活性和非活性DctD？ 5)是 磷酸化DctD必需的一个特定的N-末端结构域残基 或者其他的磷酸化作用就足够了？ 这些 研究涉及通过PCR扩增DNA、基因克隆和遗传修饰。 dctA启动子区的工程改造;过表达和纯化 DctD;以及ATP水解、转录 起始和蛋白质与DNA的结合。 结果将有助于 NIH的使命是了解细菌中的信号转导， 与毒力特性的调节和许多其他重要的 细菌活性。 它们还可以通过以下方式帮助改善固氮作用 根瘤菌

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.