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

为什么细胞会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着无数的细胞形状，它们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，一些细菌合成独特的“附属物”结构，或局部细胞延伸。例如，有柄细菌刺疣微菌在其整个表面上产生大量刺突，肺炎支原体粘附需要称为尖端结构的膜结合细胞延伸。为了研究这种细菌附属物的调控和合成，本项目以模式细菌新月柄杆菌为研究对象。初步数据表明，茎细胞壁在化学上与细胞体不同;这一发现是惊人的，因为它将是第一个证明细菌具有包含独特化学结构的差异调节结构域。对茎细胞壁的化学和机械性质的详细了解以及对参与茎伸长的蛋白质的表征将为细菌形状和区室化的进化和功能的基本问题提供重要的见解。除了本项目的研究目标外，还有几个针对高中生、本科生和研究生的教育目标。将为每个级别的学生开发新课程，以培养下一代生物学定量方法的科学家，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LEAP学院的学生，该学院90%的入学人数是代表性不足的少数民族。这些程序的目的是接口，并有助于两个研究项目。本项目的目标是确定本地化的细菌包膜合成和附属物形成的机制，使用Caulobacter crescentus的极柄作为模型系统。中心的假设是，柄杆菌产生一个极性柄，其肽聚糖（PG）具有不同的化学和机械性能的细胞体PG。以前的研究柄合成集中在已知的PG调节酶或柄相关蛋白的定位。相比之下，这种方法是创新的，因为出发点是观察到茎包膜在组成上不同于细胞体。从这个角度来看，中心假设将通过以下具体目标进行检验：1）定义茎肽聚糖的组成和机械性质; 2）有针对性地分析茎生物合成调控蛋白; 3）通过对茎表型突变体进行基因分型来鉴定新的茎伸长基因。在第一个目标下，实验方法将用于确定秸秆PG的化学组成和量化的弹性模量。PG组合物和机械性能之间的联系将通过计算建模进一步探讨。在第二个目标下，将使用新型MreB荧光融合构建体研究MreB的作用，所述新型MreB荧光融合构建体对于茎生物发生是有缺陷的，同时保留野生型细胞生长功能。对推定的转肽酶CC 2105的研究将集中在C-末端结构域，该结构域在柄杆菌科的有柄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。拟议中的研究预计将有助于深入了解如何茎特定的信封合成的结果在区室调节细胞形状。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产生亚细胞区室/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.