B SUBTILIS PHO SIGNAL TRANSDUCTION NETWORK

描述：双分量信令是信号的主要形式 原核生物中的转导与真核生物中独特的例子。 对枯草芽孢杆菌的相当大的兴趣一直集中在鉴定 启动这一过程的环境和生理信号 产孢子。与营养和细胞周期相关的信号产生在 后期生长由相互关联的调控途径处理，这些调控途径控制着 哪些基因被表达，通常最终导致 产孢子。枯草杆菌碱性变种的等电点特征 磷酸酶(APase)多基因家族导致了一种假说 不同的环境信号由特定的两个分量接收 集成传入信息以确定响应的系统 枯草杆菌对磷有效性下降的影响。 本提案旨在了解三个独立的 双组分系统(Phop-Phor、ResD-Rese和SpoOA)，每个接收 他们特定的环境信号，通过交流来实现最终的Pho 回应。PI处于幸运的地位，拥有基本的 信号转导电路的架构已就位。这提供了一种 测试和完善她的假说的框架。平行通道 通过Pho-Phor对Pho反应进行正向调节。一条途径包括 RESD-RESE系统，而另一种涉及过渡状态调节器， AbrB.Spoo系统通过负性调节抑制Pho反应 两条路都有。持续的基因研究将被用来进一步识别 光合激活途径与生化反应中的中间产物 研究计划探索它们之间的相互作用。抑制者 SpoOA在AbrB激活途径中的作用已有较好的认识。《少年派》 将使用遗传和生化技术来确定 SpoA抑制RESA启动子，这是导致 ResDE基因的转录。她将使用生化方法来 探索PHOR中的两个域，以了解可能的信号机制。推动者 已知需要用于激活的PHIP将被分析以进行直接交互 以及在这些序列中的保守序列的可能功能 推动者。 这项提议有几个阶段。第一个是利用基因 选择或筛选以识别该综合体的新组件， 相互作用的调节子。在PhoPR操纵子中有四个RNA起始点 由引物延伸确定的启动子区域，以及详细的 在不同的突变背景下对这些基因进行定量分析将确定 哪些部位受哪条途径调控。还进行了缺失分析 有计划的。 Php和Pho本身、凝胶延迟和DNA的特征 足迹试验都计划用来评估对 各种Pho调控的启动子。Phor包含两个区域，其中 与外部(两个跨膜区域之间的区域)的相互作用 和内部(第二跨膜区域和 组氨酸激活域)信号可能会出现。这些基因将发生突变或 替换为其他传感器激酶的相应结构域和 评估了诱导对磷酸盐饥饿的影响。互动中的 将使用交联法寻找蛋白质。

DESCRIPTION: Two-Component signaling is the principle form of signal transduction in procaryotes with distinctive examples in eucaryotes. Considerable interest in Bacillus subtilis has been focused on identifying the environmental and physiological signals that initiate the process of sporulation. Nutritional and cell cycle related signals produced during late growth are processed by interconnected regulatory pathways that control which genes are expressed, often culminating in the initiation of sporulation. The PI's characterization of the B. subtilis alkaline phosphatase (APase) multigene family has led to the hypothesis that different environmental signals are received by specific two-component systems that integrate the incoming information to determine the response of B. subtilis to declining phosphate availability. The present proposal is designed to understand how three separate two-component systems (PhoP-PhoR, ResD-ResE, and SpoOA), each receiving their specific environmental signals, communicate to achieve the final Pho response. The PI is in the fortunate position of having the basic architecture of the signal transduction circuitry in place. This provides a framework for testing and refining her hypothesis. Parallel pathways positively regulate the Pho response via PhoR-PhoR. One pathway includes the ResD-ResE system, while the other involves a transition state regulator, AbrB. The SpoO system represses the Pho response by negatively regulating both pathways. Continuing genetic studies will be used to further identify intermediates in the ResD to PhoP-PhoR activation pathway and biochemical studies are planned to probe the interactions between them. The repressor function of SpoOA on the AbrB activation pathway is well udnerstood. The PI will use genetic and biochemical techniques to determine the mechanism of SpoOA repression of the resA promoter which is responsible for the transcription of the resDE genes. She will use biochemical approaches to explore two domains in the PhoR for pssible signaling mechanisms. Promoters known to require PhoP for activation will be analyzed for direct interaction with PhoP and for possible function of conserved sequences within these promoters. This proposal has a number of phases. The first is to employ genetic selections or screenings to identify new components of this complex, interacting regulon. There are four RNA start sites in the phoPR operon promoter region as determined by primer extension, and a detailed quantitative analysis of these in various mutant backgrounds will determine which sites are regulated by which pathway. A deletion analysis is also planned. To characterize PhoP and PhoR themselves, gel retardation and DNA footprinting experiments are all planned to assess direct regulation on various Pho regulated promoters. PhoR contains two regions where interaction with external (the region between two membrane-spanning regions) and internal (the region between the second membrane-spanning region and the histidine kinase domain) signals could occur. These will be mutated or substituted with corresponding domains of other sensor kinases and the effect of the induction upon phosphate starvation assessed. The interacting proteins will be sought using cross-linking.