REGULATION OF NODULATION GENES IN RHIZOBIUM MELILOTI

我们的建议涉及原核生物中基因表达的调控， 苜蓿根瘤菌，感染并建立共生， 真核植物宿主，如苜蓿（Medicago sativa）。 我们有 以前建立的作用，植物诱导剂结合 包括基因激活蛋白NodD的细菌调节装置。 一种诱导剂非依赖性途径，涉及两种其他激活剂，SyrM和 NodD 3已经被发现，我们已经证明它是在复杂的控制下。 我们最近已经表明，NodD和SyrM激活剂需要以下之一： 两个groESL基因被激活 在下一个项目期间，我们将 进一步追求控制的基因，无论是在定义的 控制它的电路，并在检查的生化机制， nod启动子-NodD活性。 I.我们将开发新的遗传工具，包括转座子，载体， 条件噬菌体突变体和限制性R.苜蓿品系， 促进进一步的全球基因搜索所需的基因座节点 启动子表达 我们将完成对BRB抑制的分析 的。 我们将研究非NodD依赖性的遗传基础， 诱导剂木樨草素的促生长作用。 二.我们提出了一个模型，指导实验设计的节点 启动子表达 三.我们将绘制CIMM起始位点和其他几个启动子， 增加我们对原生启动子的了解。 我们将使用诱变技术， 选择人工站点，以确定节点D的要求， SyrM结合。 通过删除分析，我们将检查节点的作用 mRNA前导序列。 四.我们将使用一系列突变筛选，包括一些 区分完整启动子和半位点，以鉴定结构域 在NodD 3和NodD 1中，DNA结合，蛋白质-蛋白质协同性， 以及与聚合酶的相互作用。 我们将独立测试 NodD中存在二聚化结构域。 基于我们定义的 激活域内的两个NodD的，我们将问是否SyrM 蛋白质需要相同的结构域来激活RNA聚合酶。 诉我们将进一步纯化和表征RNA聚合酶， GroEL和GroES用于进一步的生物化学研究。 康贝特人将以 GroE在NodD活动中所扮演的角色，并询问它是否具有 影响NodD与RNA聚合酶的相互作用。 我们将分析 诱导剂如木犀草素对RNA相互作用的影响 聚合酶、NodD和GroEL。 六.我们将寻找导致节点减少的遗传电路 启动子在拟杆菌中的表达。 我们还将测试网站， 重要的是，这种减少，包括任何确定的领导人， 第一部分.

Our proposal concerns the regulation of gene expression in a prokaryote, Rhizobium meliloti, that infects and establishes symbiosis with eukaryotic plant hosts such as alfalfa (Medicago sativa). We have previously established a role for plant inducers in combination with a bacterial regulatory apparatus including the gene activator protein NodD. An inducer-independent pathway involving two other activators, SyrM and NodD3, has been found, and we have shown it is under complex control. We have recently shown that the NodD and SyrM activators require one of two groESL genes to be active. In the next project period, we will pursue further the control of nodulation genes, both in definition of the circuits that control it, and in examining the biochemical mechanism of nod promoter-NodD activity. I. We will develop new genetic tools including transposons, vectors, conditional phage mutants and a restriction R. meliloti strain, to facilitate further global genetic searches for loci needed for nod promoter expression. We will complete analysis of the syrB repression of syrM. We will examine the genetic basis for the NodD-independent growth-promotive effect of the inducer luteolin. II. We present a model that guides the design of experiments on nod promoter expression. III. We will map the syrM start site and several other promoters, to increase our knowledge of native promoters. We will use mutagenesis and selection of artificial sites to define the requirements for NodD and SyrM binding. By deletion analysis, we will examine the role of the nod mRNA leader sequence. IV. We will use a series of mutant screens, including some that distinguish between intact promoters and half sites, to identify domains in NodD3 and NodD1 for DNA binding, for protein-protein cooperativity, and for interaction with polymerase. We will test independently for the presence of dimerization domains in NodD. Based on our defining an activator domain within the two NodD's we will ask whether the SyrM protein requires the same domain for activation of RNA polymerase. V. We will further purify and characterize RNA polymerase and will purify GroEL and GroES for further biochemical study. We will determine the role played by GroE in the activity of NodD and ask whether it has an effect in the interactions of NodD with RNA polymerase. We will assay the effect of inducers such as luteolin on the interactions of RNA polymerase, NodD, and GroEL. VI. We will look for the genetic circuit that causes the decrease of nod promoter expression in bacteroids. We will also test for sites that are important for this decrease, including any identified from the leader in part I.