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

职业：新月柄杆菌茎的组成、机械性能和合成

• 批准号: 1553004
• 负责人: Eric Klein
• 金额: $ 123.8万
• 依托单位: Rutgers University Camden
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1553004&HistoricalAwards=false
• 关键词: CAREER; Composition; mechanical; properties; synthesis

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.

为什么细胞发育并保持特定的形状是细胞生物学的一个基本问题。对细菌细胞形状维持的了解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。确定这些不寻常形态的作用和潜在机制可以为广泛的细菌生理特性提供深刻的见解。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，被跟踪的棘疣菌（Verrucomicrobium spinosum）在其整个表面产生大量的尖刺，而肺炎支原体的粘附需要一种被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，本项目重点研究了模型跟踪细菌新月形Caulobacter crescentus。初步资料表明，茎细胞壁的化学性质与细胞体不同；这一发现是惊人的，因为它将首次证明一种细菌具有包含独特化学结构的差异调节结构域。详细了解茎细胞壁的化学和机械特性以及参与茎伸长的蛋白质特征将为了解细菌形状和区隔化的进化和功能的基本问题提供重要的见解。除了这个项目的研究目标之外，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的课程，以培养下一代科学家在生物学的定量方法，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LEAP学院的学生，该学院90%的学生是少数族裔。这些程序中的每一个都被设计为两个研究项目的接口和贡献。本项目的目的是利用新月茎杆菌的极柄作为模型系统，确定局部细菌包膜合成和附属物形成的机制。核心假设是，Caulobacter产生极性柄，其肽聚糖（PG）具有与细胞体PG不同的化学和机械特性。先前对柄合成的研究要么集中在已知的PG调节酶上，要么集中在柄相关蛋白的定位上。相比之下，这种方法是创新的，因为它的出发点是观察到茎包膜的组成不同于细胞体。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)确定秸秆肽聚糖的组成和力学性质；2)秸秆生物合成调控蛋白的靶向分析；3)通过对茎秆表型突变体进行基因分型，鉴定新的茎秆伸长基因。在第一个目标下，实验方法将用于确定秸秆PG的化学组成和量化弹性模量。PG组成与力学性能之间的联系将通过计算建模进一步探索。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，该结构在茎秆生物发生中存在缺陷，但在细胞生长中保留野生型功能。对推测的转肽酶CC2105的研究将集中在一个c端结构域，该结构域在茎杆菌科的跟踪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。拟议的研究预计将有助于深入了解茎特异性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产生亚细胞区室/附属物的一般策略。

项目成果

Sugar-Phosphate Metabolism Regulates Stationary-Phase Entry and Stalk Elongation in Caulobacter crescentus

• DOI: 10.1128/jb.00468-19
• 发表时间: 2020-02-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: de Young, Kevin D.;Stankeviciute, Gabriele;Klein, Eric A.
• 通讯作者: Klein, Eric A.

AimB Is a Small Protein Regulator of Cell Size and MreB Assembly

• DOI: 10.1016/j.bpj.2020.04.029
• 发表时间: 2020-08-04
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Werner，John N.;Shi，Handuo;Klein，Eric A.
• 通讯作者: Klein，Eric A.

Caulobacter crescentus Adapts to Phosphate Starvation by Synthesizing Anionic Glycoglycerolipids and a Novel Glycosphingolipid

• DOI: 10.1128/mbio.00107-19
• 发表时间: 2019-04
• 期刊: mBio
• 影响因子: 6.4
• 作者: Gabriele Stankeviciute;Z. Guan;H. Goldfine;Eric A. Klein

Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A

柄杆菌需要阴离子鞘脂和毛皮失活才能失去脂质 A

• DOI: 10.1101/2022.01.20.477143
• 发表时间: 2022
• 期刊: bioRxiv
• 作者: Zik, J;Yoon, S;Guan, Z;Skidmore, G;Gudoor, R;Davies, K;Deutschbauer, A;Goodlett, D;Klein, E;Ryan, K.
• 通讯作者: Ryan, K.

Purification and HPLC Analysis of Cell Wall Muropeptides from Caulobacter crescentus

新月柄杆菌细胞壁胞肽的纯化及高效液相色谱分析

• DOI: 10.21769/bioprotoc.3421
• 发表时间: 2019
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Stankeviciute, Gabriele;Klein, Eric
• 通讯作者: Klein, Eric