PodJ processing and polar development in Caulobacter crescentus

描述（由申请人提供）：本研究的长期目标是确定细菌细胞如何建立细胞内极性，将特定过程引导到极点，并控制极性过程的活性。许多细菌以细胞器和/或过程为目标，专门针对细胞极点。特别是，有研究表明，许多病原体极点定位毒力因子，表明极点定位对疾病有影响。极性定位的基本机制尚不清楚。在这项工作中，新月茎杆菌的发展被用作极性的模型系统。这种生物的极性发育与蛋白质PodJ的亚细胞定位和蛋白水解加工有关，而在发育中受到抑制的月形c.c crescentus细胞不显示PodJ加工，这表明PodJ的稳定性与极性发育过程有关。在其他生物体中也发现了类似PodJ的其他蛋白质，包括表现出细胞极性的病原体。通过了解PodJ在控制极性过程中的作用，有可能将这一知识外推到其他细菌系统。这项工作的具体目的是：1.；2.在PodJ中产生突变，导致蛋白质稳定性增加，并确定PodJ介导的极性过程稳定性增加的影响；确定假定的PodJ肽聚糖结合域在维持PodJ稳定性中的作用。将结合预测影响PodJ稳定性的序列的定点突变，以及使用强大的测定方法鉴定PodJ稳定性突变体的无偏遗传筛选。突变表型的分析将涉及细胞生物学技术、生物化学技术和细菌生理学分析。一些植物和动物病原体表现出细胞极性和毒力因子的极性定位，但细菌极性的基本机制尚不清楚。了解建立和控制细菌极性的基本方法将有助于更容易地获得病原体采用的特定策略，并增强对抗其毒性的新策略。

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine how bacterial cells establish intracellular polarity, direct specific processes to the poles, and control the activity of polar processes. Many bacteria target organelles and/or processes specifically to the cell poles. Particularly, it has been shown that many pathogens polarly localize virulence factors, indicating that polar localization has implications in disease. The basic mechanisms for polar localization are unknown. In this work, development of Caulobacter crescentus is used as a model system for polarity. Polar development in this organism has been correlated with the subcellular localization and proteolytic processing of the protein PodJ, and C. crescentus cells that are inhibited in development show no PodJ processing, indicating that the stability of PodJ is tied to polar developmental processes. Other proteins similar to PodJ have been found in other organisms, including pathogens that demonstrate cellular polarity. By understanding the role of PodJ in controlling polar processes, it may be possible to extrapolate this knowledge to other bacterial systems. The specific aims of this work are: 1. generate mutations in PodJ that lead to increased protein stability and determine the affect of increased stability of PodJ-mediated polar processes, and 2. determine the role of the putative PodJ peptidoglycan binding domain in maintaining PodJ stability. A combination of site-directed mutagenesis of sequences predicted to impact PodJ stability, and unbiased genetic screens using powerful assays to identify PodJ stability mutants will be utilized. Analysis of mutant phenotypes will involve cell biology techniques, biochemistry techniques, and bacterial physiology assays. Several plant and animal pathogens demonstrate cellular polarity and polar localization of virulence factors, yet the basic mechanisms of bacterial polarity are unknown. Understanding the underlying methods of establishing and controlling bacterial polarity will facilitate easier access to specific strategies employed by pathogens, and enhance new strategies to combat their virulence.