CAREER: Composition, mechanical properties, and synthesis of the Caulobacter crescentus stalk

细胞为什么发育并保持特定的形状是细胞生物学中的一个基本问题。了解细菌细胞形状的维持主要集中在杆状细胞上。然而，我们知道自然界中存在的无数细胞形状是由强大的选择压力维持的。确定这些不寻常的形态的作用和潜在机制可以提供对广泛的细菌生理特性的深入了解。特别是，一些细菌会合成独特的“附属物”结构，或局部的细胞延伸。例如，茎杆细菌Verrucommicrobium Spinosum在其整个表面产生大量的尖刺，肺炎支原体的粘连需要一个称为尖端结构的膜结合细胞延伸。为了研究这类细菌附属物的调控和合成，本项目以模式菌柄新月形杆菌为研究对象。初步数据表明，柄细胞壁在化学上与细胞体的细胞壁不同；这一发现令人震惊，因为这将是第一次证明细菌具有包含独特化学结构的不同调控结构域。详细了解茎细胞壁的化学和机械性质以及参与茎伸长的蛋白质的特征将为了解细菌形态和区划的进化和功能这一基本问题提供重要的见解。除了这个项目的研究目标外，还有几个针对高中、本科生和研究生的教育目标。将为每个级别的学生开发新的课程，以培训下一代科学家在生物学中的定量方法，包括生物信息学、定量图像分析和生物物理学。特别是，高中部分将涉及卡姆登的LEAP学院的学生，他们的入学率90%是代表不足的少数族裔。这些程序中的每一个都被设计为与这两个研究项目相结合并做出贡献。该项目的目标是以新月弯杆菌的极柄为模型系统来确定局部细菌包膜合成和附属物形成的机制。中心假说是，Caulbacter产生一种极性茎，其肽聚糖(PG)具有与细胞体PG不同的化学和机械性质。以往对秸秆合成的研究要么集中在已知的PG调节酶上，要么集中在秸秆相关蛋白的定位上。相反，这种方法是创新的，因为起点是观察到茎被膜的成分与细胞体的成分不同。从这一角度来看，中心假说将通过以下具体目标得到验证：1)确定秸秆肽聚糖的组成和机械性质；2)有针对性地分析秸秆生物合成调节蛋白；以及3)通过对茎表型突变进行基因分型来发现新的茎伸长基因。在第一个目标下，将采用实验方法来确定秸秆PG的化学组成并量化其弹性模数。PG的组成和力学性能之间的联系将通过计算模型进一步探索。在第二个目标下，将使用一种新的MreB荧光融合结构来研究MreB的作用，这种结构对茎的生物发生有缺陷，同时保留了野生型的细胞生长功能。对可能的转肽酶CC2105的研究将集中在一个C-末端结构域，该结构域在硫杆菌科细菌中是唯一保守的。在第三个目标中，LEAP学院的高中生将使用传统遗传学方法和下一代测序相结合的方法来表征一组最近分离的茎伸长突变体。这项拟议的研究预计将有助于深入了解茎特定的包膜合成如何导致细胞形状的区隔调节。细菌细胞生物学领域的这一垂直进展表明，局部形态的动态调节可能是细菌维持对细胞形状的生理控制或创建亚细胞隔间/附属物的一般策略。

Why cells develop and maintain a particular shape represents a fundamental question in cell biology. Understanding the maintenance of bacterial cell shape have primarily focused on rod-shaped cells. Yet we know of myriad cell shapes present in nature that are maintained by strong selective pressures. Determining the roles and underlying mechanisms of these unusual morphologies can provide great insight into a wide range of bacterial physiological properties. In particular, some bacteria synthesize unique "appendage" structures, or localized cellular extensions. For example, the stalked bacterium Verrucomicrobium spinosum produces a large number of spikes across its entire surface and Mycoplasma pneumoniae adherence requires a membrane-bound cell extension termed the tip-structure. In order to study the regulation and synthesis of such bacterial appendages, this project focuses on the model stalked bacterium Caulobacter crescentus. Preliminary data suggest that the stalk cell wall is chemically distinct from that of the cell body; this finding is striking because it would be the first demonstration of a bacterium with differentially regulated domains containing unique chemical structures. A detailed understanding of the chemical and mechanical properties of the stalk cell wall and characterization of proteins involved in stalk elongation will provide important insight into the fundamental question of the evolution and function of bacterial shape and compartmentalization. In addition to the research goals of this project, there are several educational objectives targeting high school, undergraduate, and graduate students. New courses will be developed for students at each level to train the next generation of scientists in quantitative methods in biology including bioinformatics, quantitative image analysis, and biophysics. In particular, the high school component will involve students at the LEAP Academy in Camden, whose enrollment is 90% underrepresented minorities. Each of these programs is designed to interface with and contribute to the two research projects.The objective of this project is to determine the mechanisms underlying localized bacterial envelope synthesis and appendage formation using the polar-stalk of Caulobacter crescentus as a model system. The central hypothesis is that Caulobacter produces a polar stalk whose peptidoglycan (PG) has distinct chemical and mechanical properties from the cell body PG. Previous studies of stalk synthesis have either focused on known PG regulating enzymes or on the localization of stalk associated proteins. In contrast, this approach is innovative because the starting point is the observation that the stalk envelope is compositionally different than that of the cell body. From this perspective, the centralhypothesis will be tested by the following specific aims: 1) Define the composition and mechanical properties of stalk peptidoglycan; 2) Targeted analysis of the stalk biosynthesis regulatory proteins; and 3) Identify novel stalk-elongation genes by genotyping stalk-phenotype mutants. Under the first aim, experimental approaches will be used to determine the chemical makeup and quantify the elastic modulus of stalk PG. The link between PG composition and mechanical properties will be further explored via computational modeling. Under the second aim, the role of MreB will be investigated using a novel MreB fluorescent fusion construct that is defective for stalk biogenesis while retaining wild type function for cell growth. Studies of the putative transpeptidase CC2105 will focus on a C-terminal domain which is uniquely conserved among the stalked bacteria of the Caulobacteraceae family. In the third aim, high school students at the LEAP Academy will use a combination of traditional genetics methods and next generation sequencing to characterize a set of recently isolated stalk-elongation mutants. The proposed research is anticipated to contribute in-depth knowledge of how stalk-specific envelope synthesis results in compartmentalized regulation of cell shape. This vertical advance in the field of bacterial cell biology suggests that the dynamic regulation of localized morphology may be a general strategy employed by bacteria to maintain physiological control over cell shape or create subcellular compartments/appendages.