Characterization of non-canonical regulatory pathways in the Caulobacter NtrYX signaling system

项目总结/摘要 所有的细菌都必须感知并响应代谢和环境的变化，才能在多样化的环境中茁壮成长。 栖息地这些自适应响应通常使用双分量信号系统来实现。二-- 组成信号系统由两个核心要素：感觉组氨酸激酶和反应 调节器组氨酸激酶检测到一个信号并磷酸化反应调节因子。总的来说， 反应调节剂的磷酸化导致基因表达的变化。虽然典型的双组分 虽然信令系统有些简单，但越来越明显的是，许多系统更加复杂。 复杂的感觉网络，包含非典型特征和额外的调节成分。NtrY-NtrX （NtrYX）是一个新兴的例子，一个复杂的感觉系统与不同的表型，在许多疾病， 变形菌先前的研究已经在规范双组分的框架内研究了NtrYX 发信号。然而，最近的研究表明，在淡水和土壤细菌新月柄杆菌中， NtrYX系统更复杂，包含一个未知的磷酸化来源和一个新的NtrY 调节器，NtrZ。此外，遗传和转录数据表明，磷酸化和非磷酸化的 NtrX具有独特的生理相关活性。拟议的工作将调查综合监管 策略在C. crescentus NtrYX系统在多个层面。该项目的第一个目标将决定如何 NtrX磷酸化影响其DNA结合和调节活性。为了更好地理解 第二个目标将应用生物化学和结构方法来探测磷酸化的水平。 NtrY和NtrZ调节器之间的相互作用。最后，第三个目标将探索一条额外的途径 调节磷酸流，采用候选人驱动和无偏倚的方法来鉴定体内来源 NtrX的磷酸化。总之，这些方法将建立C。crescentus NtrYX作为一个强大的模型， 了解交叉调节途径如何控制双组分信号传导。我们的工作也将 揭示了保守的和重要的NtrYX系统在不同的变形菌，包括致病性和 共生微生物如流产布鲁氏菌和苜蓿中华根瘤菌。此外，该项目将揭示新颖， 用于调谐细菌中的信号转导途径的可推广策略。

Project Summary/Abstract All bacteria must sense and respond to metabolic and environmental changes to thrive in diverse habitats. These adaptive responses are often achieved using two-component signaling systems. Two- component signaling systems are composed of two core elements: a sensory histidine kinase and response regulator. The histidine kinase detects a cue and phosphorylates the response regulator. In general, phosphorylation of response regulators leads to changes in gene expression. Although canonical two-component signaling systems are somewhat simple, it has become increasingly apparent that many systems are more complex sensory networks, incorporating atypical features and additional regulatory components. NtrY-NtrX (NtrYX) is an emerging example of a complex sensory system linked to diverse phenotypes in many ⍺- proteobacteria. Prior studies have investigated NtrYX within the framework of canonical two-component signaling. However, recent work reveals that, in the freshwater and soil bacterium Caulobacter crescentus, the NtrYX system is more elaborate, incorporating an unidentified source of phosphorylation and a novel NtrY regulator, NtrZ. In addition, genetic and transcriptional data suggest that phosphorylated and unphosphorylated NtrX have distinct and physiologically relevant activities. The proposed work will investigate integrated regulatory strategies in the C. crescentus NtrYX system at multiple levels. The first aim of this project will determine how NtrX phosphorylation affects its DNA-binding and regulatory activities. To better understand how this phosphorylation is controlled, the second aim will apply biochemical and structural approaches to probe the interaction between NtrY and the NtrZ regulator. Finally, the third aim will explore an additional pathway regulating phosphoryl-flow, employing candidate-driven and unbiased approaches to identify the in vivo source of NtrX phosphorylation. Together, these approaches will establish C. crescentus NtrYX as a powerful model for understanding how intersecting regulatory pathways can control two-component signaling. Our work will also shed light on conserved and important NtrYX systems in diverse ⍺-proteobacteria, including pathogenic and symbiotic organisms like Brucella abortus and Sinorhizobium meliloti. Moreover, this project will reveal novel and generalizable strategies for tuning signal transduction pathways in bacteria.