Molecular Genetics of Bacillus subtilis RNA polymerase

描述(申请人提供)：细菌暴露在不同的生长限制压力下会诱导大量蛋白质的合成，这些蛋白质可以保护细胞免受未来可能致命的压力。这种一般的应激反应带来了一种特殊的生理状态，这种状态可以提高在自然环境、食物和某些病原相互作用中的存活率。在枯草芽孢杆菌和相关的革兰氏阳性病原体中，这种反应是由？B转录因子控制的。？B功能的丧失会导致对多种应激的敏感性增加，包括酸、抗生素、冷、热、渗透和氧化应激。我们的长期目标是以枯草杆菌为模型，从检测不同胁迫的传感器开始，延伸到将这一信息传递到？B的信号转导网络，最后到？B控制下的基因的生理作用。这项建议涉及信号网络本身，它通过“伙伴转换”机制发挥作用，在该机制中，关键的蛋白质相互作用由丝氨酸或苏氨酸磷酸化控制。这种机制似乎很古老，非常可塑性，在真细菌中很普遍。因此，对枯草杆菌中这种机制的研究应该有助于理解支配广泛信号通路的原理。在这里，它激活？B以响应两类压力：(I)能量压力，包括碳、磷或氧的饥饿；(Ii)环境压力，包括酸、乙醇、热或盐。这两个类通过不同的信号通路被传递到？B，每个信号通路的末端都有一个不同调节的PP2C磷酸酶，每个信号通路都汇聚在？B的直接调节子上，RsbV是抗？和RsbW反？，它们构成一个伙伴交换模块。能量分支由RSBP磷酸酶(具有一个对信号重要的PAS结构域)和RsbQ组成，RsbQ是一种预测的水解酶，也是必需的。环境分支有八个调节器，其中一些是多余的，所有调节器都通过第二个非典型的伙伴切换模块来激活RsbU磷酸酶。这些调节剂中的六个在一个大的(700 KDa)复合体中共同从细胞提取物中提纯，三个形成一个简化的核心，可以在体外和体内进行分析。我们的具体目标使用遗传、生化和结构相结合的方法来解决三个问题：(1)能量信号的机制是什么；(2)环境信号的机制是什么；以及(3)正反馈和负反馈在B网络中的作用是什么？

DESCRIPTION (provided by applicant): Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a large set of proteins that protect the cell against future, potentially lethal stresses. This general stress response brings about a special physiological state which enhances survival in the natural environment, in foods, and in some pathogenic interactions. Among Bacillus subtilis and related Gram positive pathogens, this response is governed by the ?B transcription factor. Loss of ?B function causes increased sensitivity to multiple stresses, including acid, antibiotic, cold, heat, osmotic, and oxidative stress. Our long term objective is to understand this response using B. subtilis as a model, beginning with the sensors which detect the different stresses, extending through the signal transduction network which conveys this information to ?B, and ending with the physiological role of genes under ?B control. This proposal addresses the signaling network itself, which functions by the "partner switching" mechanism in which key protein interactions are controlled by serine or threonine phosphorylation. This mechanism appears to be ancient, very plastic, and widespread among eubacteria. Study of this mechanism in B. subtilis should therefore help understand principles governing a broad array of signaling pathways. Here it activates ?B in response to two classes of stresses: (i) energy stress, including starvation for carbon, phosphate, or oxygen; and (ii) environmental stress, including acid, ethanol, heat, or salt. These two classes are conveyed to ?B by separate signaling pathways, each terminating with a differentially regulated PP2C phosphatase and each converging on the direct regulators of ?B, the RsbV anti-anti-? and the RsbW anti-?, which form one partner switching module. The energy branch consists of the RsbP phosphatase (with a PAS domain important for signaling) and RsbQ, a predicted hydrolase that is also required. The environmental branch has eight regulators, some redundant, and all join to activate the RsbU phosphatase via a second, atypical partner switching module. Six of these regulators co-purify from cell extracts in a large (>700 kDa) complex, and three form a simplified core amenable to analysis in vitro and in vivo. Our specific aims use a combined genetic, biochemical and structural approach to address three questions: (1) What is the mechanism of energy signaling; (2) What is the mechanism of environmental signaling; and (3) What is the role of positive and negative feedback in the ?B network?