Function & Regulation of the Bradyrhizobium japonicum nolA Gene

Stacey 9728281最近的研究已经确定了几个细菌全局调节因子的大家族。虽然这样的全球监管机构正在被广泛研究，相对知之甚少的是如何这样的监管系统可以相互作用，以整合细胞对环境的反应，和几个模型系统已被确定为研究这样的综合反应。结瘤（nod）基因在慢生型大豆根瘤菌中的表达受转录调控蛋白LysR、双组分和MerR家族成员的调控。因此，B. japonicum代表了一个有趣和有用的系统，在其中探索这些全球调节系统之间的相互作用。在B。在植物产生的异黄酮存在下，nod基因激活需要LysR同源物NodD 1。NodV和NodW组成第二个独立的系统，在添加NodV诱导剂后激活nod基因表达。最后，作为MerR家族的一员，NoIA间接抑制nod基因的表达，并可能激活其他基因的表达。本研究主要集中在nolA基因的调控和功能。令人惊讶的是，最近的结果表明，nolA基因编码三种不同的蛋白质（称为NolAL，NolAM和NolAs），其中至少两种是由不同的信使RNA合成的。此外，nolA的表达似乎是由一个独特的植物信号分子激活。已经构建了定点突变，在每种情况下，导致仅一种形式的NoIA的表达。使用这些构建体，三种NolA蛋白的独立调节正在研究中。将确定NolA的三种形式中的哪一种在确定大豆基因型之间的共生宿主范围中是重要的。nolA基因从至少两个独立的转录起始位点（P1和P2）表达。从P1表达似乎需要新的植物信号分子，而从P2表达需要功能性NoIA。将构建P1和P2两者的5'区域中的突变，以在竞争模型之间测试如何介导它们的独立调节。NoIAM和NoIAS的表达可能是由于第三个未识别的转录起始位点，或由于转录后调控事件。此外，将确定ATG 2（NolAM的起始）附近的二级结构在确定NolAM和NolAS的相对表达中可能是重要的可能性。 最后，将完成诱导nolA表达的来自大豆植物的独特化学品的纯化。这些研究将阐明NolA在nod基因表达中的作用。所获得的结果，当添加到其他正在进行的研究，应该给B的nod基因表达的一个更完整的图片。并且可以作为其他复杂调控系统的范例，其中全球调控网络影响单个操纵子的表达。B。大豆-大豆共生系统是研究寄主与共生体之间细胞信号传导途径的重要系统。这项工作的最终目标是更清楚地了解参与细胞代谢全球调节的调节机制的整合。

Stacey 9728281 Recent research has identified several large families of bacterial global regulators. Although such global regulators are being extensively studied, relatively little is known about how such regulatory systems may interact to integrate the cellular response to the environment, and few model systems have been identified to study such integrated responses. Nodulation (nod) gene expression in Bradyrhizobium japonicum is regulated by members of the LysR, two-component, and MerR families of transcriptional regulatory proteins. Therefore, it is possible that regulation of nod gene expression in B. japonicum represents an interesting and useful system in which to explore the interactions between these global regulatory systems. In B. japonicum, a LysR homologue, NodD1, is required for nod gene activation in the presence of plant produced isoflavones. NodV and NodW make up a second, independent system that activates nod gene expression upon addition of the isoflavone inducer. Finally, NoIA, a member of the MerR family, acts indirectly to repress nod gene expression and likely activates the expression of other genes. This research focuses primarily on the regulation and function of the nolA gene. Surprisingly, recent results indicate that the nolA gene encodes three separate proteins (termed NolAL, NolAM, and NolAs ), at least two of which are synthesized from separate messenger RNAs. Moreover, nolA expression appears to be activated by a unique plant signal molecule. Site directed mutations have been constructed that, in each case, result in the expression of only one form of NoIA. Using these constructs, the independent regulation of the three NolA proteins is being investigated. Which of the three forms of NolA is important in determining symbiotic host range among genotypes of soybean will be determined. The nolA gene is expressed from at least two independent transcriptional start sites (P1 and P2). Expression from P1 appears to require a novel plant signal molecule, while expressi on from P2 requires a functional NoIA. Mutations in the 5' region of both P1 and P2 will be constructed to test between competing models of how their independent regulation may be mediated. Expression of NoIAM and NoIAS may be due to a third, unidentified transcriptional start site, or due to a posttranscriptional regulatory event. In addition, the possibility that secondary structure in the vicinity of ATG2 (initiation of NolAM) may be important in determining the relative expression of NolAM and NolAS will be determined. Finally, purification of the unique chemical from soybean plants that induces nolA expression will be done. These studies should clarify the role of NolA in nod gene expression. The results obtained, when added to other on-going research, should give a more complete picture of nod gene expression in B. japonicum and may serve as a paradigm for other complex regulatory systems where global regulatory networks impinge on the expression of a single operon. The B. japonicum-soybean symbiosis is an important system for exploring the cell signaling pathways between host and symbiont. The ultimate goal of this work is a clearer understanding of the integration of regulatory mechanisms involved in the global regulation of cellular metabolism.