Caulobacter cell shape and cytoskeletal regulation

描述（由申请人提供）：所有细胞都有分子机制，确保它们形成具有选择适应性优势的特征形状。例如，许多细菌以特定的空间和时间模式组装肽聚糖细胞壁，形成杆状，促进运动和宿主入侵。介导细菌细胞形状决定的蛋白质大部分是已知的，有大量证据表明这些肽聚糖组装和模式蛋白必须受到高度调节。例如，不同的物种调节相同的保守形状决定因素，以建立不同的形状，如弯曲或螺旋杆。即使在单个细胞内，细胞形状决定机制在细胞周期的不同阶段和不同的代谢状态下也有不同的功能。尽管调节细胞形状具有重要意义，但人们对不同环境下调节肽聚糖组装蛋白的机制知之甚少。为了理解细胞形状蛋白的调控，我们正在利用新月形茎杆菌的独特特征，这是一种革兰氏阴性弯曲杆，具有易于同步的细胞周期和丰富的遗传和基因组工具包。我们提出三个独立的目标来解剖细胞形状调节。首先，我们将仔细定量MreBCD、PBP1、PBP2和RodAZ细胞延伸蛋白的动态。我们将确定这些动力学是如何相互调节的，通过细胞周期进程，并通过已知的细胞周期调节剂。我们还将在曲率突变体中成像延伸蛋白动力学，以了解延伸和曲率机械如何相互关联。其次，我们将使用我们之前开发的高通量筛选工具以及体内和体外MreB组装分析来识别和表征新的MreB调节因子。这些努力将包括表征我们最近发现的两种MreB调节因子MbiA和AimB的作用机制，以及鉴定调节MreB的新蛋白。第三，我们将利用我们最近发现的CtpS作为双功能代谢酶和细胞形状调节剂来定义形状和代谢是如何共同调节的。具体来说，我们将确定代谢物水平如何影响CtpS的组装和形状，CtpS如何与另一种曲率决定聚合物新月素相互作用，以及将形状与代谢状态耦合的选择性益处。我们的努力将首次全面了解细胞生长如何在不同状态下受到调节，以及它如何与其他细胞过程（如分裂和代谢）耦合。这些研究将为其他系统的细胞形状研究提供一个路线图。此外，决定形状的细菌细胞壁是抗生素药物最常见的靶标之一。因此，我们的工作将有助于开发未来几代抗生素，以补充由于对现有药物的耐药性上升而迅速耗尽的治疗武器库。

DESCRIPTION (provided by applicant): All cells have molecular mechanisms that ensure that they develop characteristic shapes with selective fitness advantages. For example, many bacteria assemble peptidoglycan cell walls in specific spatial and temporal patterns to take on rod-like shapes that promote motility and host invasion. The proteins that mediate bacterial cell-shape determination are largely known and there is substantial evidence that these peptidoglycan assembly and patterning proteins must be highly regulated. For example, different species modulate the same conserved shape determinants to establish different shapes, such as curved or helical rods. Even within a single cell, the cell shape determination machinery functions differently at different stages of the cell cycle and in different metabolic states. Despite the central importance of regulating cell shape, very little is known about the mechanisms that regulate peptidoglycan assembly proteins in different contexts. To understand the regulation of cell shape proteins, we are harnessing the unique features of Caulobacter crescentus, a gram-negative curved rod with a readily synchronized cell cycle and rich genetic and genomic toolkit. We propose three independent aims to dissect cell shape regulation. First, we will carefully quantitate the dynamics of the MreBCD, PBP1, PBP2, and RodAZ cell elongation proteins. We will determine how these dynamics are regulated by one another, by cell cycle progression, and by known cell cycle regulators. We will also image elongation protein dynamics in curvature mutants to understand how the elongation and curvature machineries inter-relate. Second, we will use our previously developed high-throughput screening tools and both in vivo and in vitro assays for MreB assembly to identify and characterize novel MreB regulators. These efforts will include characterizing the mechanisms of action of two MreB regulators that we recently identified, MbiA and AimB, as well as identifying novel proteins that modulate MreB. Third, we will exploit our recent discovery of CtpS as a bifunctional metabolic enzyme and cell shape regulator to define how shape and metabolism are co-regulated. Specifically, we will determine how metabolite levels influence both CtpS assembly and shape, how CtpS interacts with another curvature-determining polymer, crescentin, and the selective benefits of coupling shape to metabolic state. Our efforts will establish the first comprehensive understanding of how cell growth is regulated in different states and how it is coupled to other cellular processes such as division and metabolism. These studies will provide a roadmap for cell shape studies in other systems. Furthermore, the shape-determining bacterial cell wall represents one of the most common targets for antibiotic drugs. Thus, our work will aid in the development of future generations of antibiotics to replenish the therapeutic arsenal that is being rapidly depleted by the rise of resistance to existing drugs.