Formation and function of cell curvature in Vibrio cholerae

弯曲的细菌代表了最常见的细菌形状之一，但其机制是 细菌变得弯曲，并且弯曲的功能在很大程度上是未知的。此外，大型聚合物 在细胞质和周质中组装，但只有细胞质聚合物被很好地表征 迄今这是一个重要的区别，因为周质蛋白必须与不同的分子竞争。 环境如氧化条件和缺乏ATP/GTP能源。我的实验室最近证明 两种蛋白质CrvA和CrvB组装成周质结构，这是建立细胞所必需的， 弯曲的病原体霍乱弧菌。在这里，我们建议利用这个系统来解决该领域的 在理解弯曲形状和周质聚合物形成方面存在差距。 在以前的工作中，我们确定和特征的第一曲率行列式的V. cholesterol，CrvA。 CrvA在周质中形成聚合物，这些聚合物形成新细胞壁的插入模式，从而导致这些细菌 曲线最近，我们发现了第二个曲率行列式，CrvB。CrvA和CrvB共定位于V. 霍乱周质，以及CrvA和CrvB共表达足以诱导直弧菌弯曲 种E.大肠杆菌、铜绿假单胞菌，甚至远亲的C. crescentus和A.根瘤菌 在这里，我们试图通过回答三个问题来更好地理解细菌曲率的决定和功能。 三个目标中的突出问题。1)CrvA/B如何在周质中组装，其氧化条件 和缺乏ATP/GTP使其成为一个非常不同的环境，从细胞质中， 细胞骨架组装？AIM 1将通过结合电子和荧光显微镜来回答这个问题 以确定CrvA/CrvB组件的结构和动力学。2)CrvAB实际上是如何产生细胞的 曲率？CrvAB与先前表征的形状决定子的不同之处在于其是周质的， 像MreB这样的其他形状图案元素的自主功能。因此，目标2将解决CrvA和 CrvB通过识别和表征它们与细胞壁和其他伴侣的相互作用来诱导曲率。 3)曲率如何影响细菌的行为？目标3将利用我们的能力， 并使用单细胞成像来确定曲率如何影响凝胶、生物膜和表面上的运动性。 这些实验将剖析由细胞弯曲导致的重要行为的生物物理基础。 这些研究将使霍乱弧菌CrvAB成为一个研究细胞生物学的强有力的模型系统 形状跨越多个尺度，回答基本问题，以推进几个领域。一是在 在分子尺度上，我们将学习聚合物如何在周质中形成。第二，在细胞尺度上，我们将学习 两种蛋白质是如何改变一系列细菌的第三，在行为/进化尺度上，我们 将学习最常见的细菌形状之一所带来的好处。

Curved bacteria represent one of the most common of bacterial shapes yet the mechanisms by which bacteria become curved and the functions of curvature are largely unknown. In addition, large-scale polymers assemble in both the cytoplasm and periplasm yet only the cytoplasmic polymers have been well characterized to date. This is an important distinction, as periplasmic proteins must contend with different molecular environments like oxidizing conditions and a lack of ATP/GTP energy sources. My lab recently demonstrated that two proteins, CrvA and CrvB, assemble into periplasmic structures that are essential for establishing cell curvature in the pathogen, Vibrio cholerae. Here we propose to leverage this system to address the field’s gaps in understanding both curved shapes and periplasmic polymer formation. In previous work, we identified and characterized the first curvature determinant in V. cholerae, CrvA. CrvA forms polymers in the periplasm that pattern the insertion of new cell wall to cause these bacteria to curve. More recently we discovered a second curvature determinant, CrvB. CrvA and CrvB colocalize in the V. cholerae periplasm, and CrvA and CrvB co-expression are sufficient to induce curvature in straight Vibrio species, E. coli, P. aeruginosa, and even distantly-related C. crescentus and A. tumefaciens. Here we seek to better understand bacterial curvature determination and function by answering three outstanding questions in three aims. 1) How do CrvA/B assemble in the periplasm, whose oxidizing conditions and lack of ATP/GTP make it a very different environment from the cytoplasm where well-characterized cytoskeletons assemble? Aim 1 will answer this question by combining electron and fluorescence microscopy to determine the structure and dynamics of CrvA/CrvB assembly. 2) How do CrvAB actually generate cell curvature? CrvAB are distinct from previously-characterized shape determinants in both being periplasmic and functioning autonomously of other shape-patterning elements like MreB. Aim 2 will thus address how CrvA and CrvB induce curvature by identifying and characterizing their interactions with the cell wall and other partners. 3) How does curvature affect bacterial behaviors? Aim 3 will harness our ability to synthetically curve bacteria and use single-cell imaging to determine how curvature affects motility in gels, biofilms, and on surfaces. These experiments will dissect the biophysical basis of important behaviors that result from cell curvature. Together these studies will establish V. cholerae CrvAB as a powerful model system for studying cell shape across multiple scales, answering fundamental questions to advance several fields. First, at the molecular scale, we will learn how polymers form in the periplasm. Second, at the cellular scale, we will learn how two proteins can curve an immense range of bacteria. And third, at the behavioral/evolutionary scale, we will learn the benefits conferred by one of the most common of bacterial shapes.