REGULATION OF NODULATION GENES IN RHIZOBIUM MELILOTI

我们的建议涉及到原核生物中基因表达的调控， 紫花苜蓿根瘤菌，感染并与之共生 真核植物寄主，如紫花苜蓿。我们有 之前建立了植物诱导剂与 细菌调节装置包括基因激活蛋白Nodd。 涉及另外两个激活剂SyrM和SyrM的非诱导剂依赖的途径 NodD3，已经被发现，我们已经证明它处于复杂的控制之下。 我们最近已经表明，Nodd和SyrM激活器需要以下之一 两个GROESL基因被激活。在下一个项目期间，我们将 进一步寻求对结瘤基因的控制，既在定义上 控制它的电路，并在检查生物化学机制 结节启动子-结节活性。 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一株限制的苜蓿根瘤菌 促进进一步的全球基因搜索，寻找NOD所需的基因座 启动子表达。我们将完成对syrB抑制的分析 SyrM。我们将研究Nodd非依赖性的遗传基础 诱导剂木犀草素的促生长作用。 第二，我们提出了一个用于指导节点实验设计的模型 启动子表达。 我们将syrM起始位置和其他几个启动子映射到 增加我们对本土推动者的了解。我们将使用诱变和 选择人工场地以确定Nodd和 SyrM绑定。通过删除分析，我们将检查节点的角色 信使核糖核酸前导序列。 IV.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个启动子，以识别结构域 在NodD3和NodD1中，对于DNA结合，对于蛋白质-蛋白质协同作用， 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 两个节点内的激活器域我们将询问SyrM 蛋白质需要相同的结构域才能激活RNA聚合酶。 五、我们将进一步纯化和鉴定RNA聚合酶，并将纯化 GroEL和Groes为进一步的生化研究奠定了基础。我们将确定 Groe在Nodd的活动中扮演的角色，并询问它是否有 NodD与RNA聚合酶相互作用的影响。我们将进行化验 木犀草素等诱导剂对RNA相互作用的影响 聚合酶、NodD和GroEL。 我们将寻找导致结节减少的遗传回路 启动子在类杆菌中的表达。我们还将测试符合以下条件的站点 对这种减少很重要，包括从领导者那里发现的任何 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.