Flavobacterium Gliding Motility: Role of Cell-Surface Components

黄杆菌滑动运动：细胞表面成分的作用

• 批准号: 0641366
• 负责人: Mark McBride
• 金额: --
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0641366&HistoricalAwards=false
• 关键词: Flavobacterium; Gliding; Motility; Role; Cell

Intellectual merit:Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. The mechanism of this form of movement has been a mystery for nearly a century. Recently 12 Gld (for "gliding") proteins required for gliding were identified. Cell-surface proteins are expected to be part of the gliding machinery but surprisingly, none of the Gld proteins appear to be exposed on the cell surface. The genetic approaches used to identify the Gld proteins would have missed redundant motility proteins with overlapping functions. A screen for transposon-induced mutants with partial defects in motility identified new mutations, termed sprA, sprB, sprC, and sprD, which appear to encode some of the missing surface components of the gliding machinery. These proteins may be adhesins that are propelled along the cell surface by the Gld proteins. Cell-surface fibrils were observed on wild-type cells but were lacking on cells of a mutant deficient for sprB, sprC, and sprD. This suggests that the fibrils are composed of Spr proteins and are part of the motility machinery. The specific aims of this project are to: (1) identify and characterize the cell-surface proteins of the motility machinery; (2) visualize the gliding motility apparatus; and (3) identify moving components of the gliding machinery. Molecular experiments will be used to characterize SprA, SprB, SprC, and SprD, and novel genetic approaches will be used to identify additional cell-surface components of the motility machinery. Modern electron microscopy (EM) approaches, including cryo-EM tomography and immuno-EM, will be used to visualize the motility apparatus. The roles of the Spr proteins will be probed by determining the effect of specific antibodies on motility, and by fluorescently labeling epitope-tagged Spr proteins to visualize dynamic changes that occur during cell movement. These studies will elucidate the mechanism of a novel yet common form of bacterial motility and will result in new information regarding molecular motors, transporters, and adhesins.Broader Impacts:Undergraduate and graduate students will conduct much of this research. These students will gain experience in genetics, molecular biology, and modern microscopic approaches to study questions of bacterial cell biology. Research on F. johnsoniae will also be incorporated into several undergraduate laboratory courses. From past experience, participation of students in real research where the outcome is not known beforehand (even by the instructor) ignites scientific curiosity and appreciation of the scientific method in a way that "cookbook" laboratory exercises cannot. Genetic tools developed for F. johnsoniae and related bacteria within the phylum Bacteroidetes will be made available to the scientific community and the results of the research will be reported by students at national and international meetings and published in leading journals.

智力优势：约氏黄杆菌的细胞通过一种称为滑动运动的过程在表面上快速移动。近世纪来，这种运动形式的机理一直是个谜。最近，鉴定了12种滑行所需的Gld（用于“滑行”）蛋白。预期细胞表面蛋白质是滑行机制的一部分，但令人惊讶的是，没有Gld蛋白质似乎暴露在细胞表面上。用于鉴定Gld蛋白的遗传方法会遗漏具有重叠功能的冗余运动蛋白。筛选转座子诱导的运动性部分缺陷的突变体发现了新的突变，称为sprA，sprB，sprC和sprD，它们似乎编码一些缺失的表面成分的滑行机器。这些蛋白质可以是由Gld蛋白质沿细胞表面沿着推进的粘附素。 在野生型细胞上观察到细胞表面原纤维，但在缺乏sprB、sprC和sprD的突变体细胞上观察到细胞表面原纤维。这表明原纤维由Spr蛋白组成，是运动机制的一部分。该项目的具体目标是：（1）识别和表征运动机制的细胞表面蛋白;（2）可视化滑行运动装置;（3）识别滑行机制的运动组件。 分子实验将被用来表征SprA，SprB，SprC和SprD，和新的遗传方法将被用来确定额外的细胞表面组件的运动机制。现代电子显微镜（EM）的方法，包括冷冻EM断层扫描和免疫EM，将被用来可视化的运动装置。Spr蛋白的作用将通过确定特异性抗体对运动性的影响，以及通过荧光标记表位标记的Spr蛋白来可视化细胞运动过程中发生的动态变化来探测。这些研究将阐明一种新的但常见的细菌运动形式的机制，并将导致有关分子马达，转运蛋白和粘附素的新信息。更广泛的影响：本科生和研究生将进行大部分研究。这些学生将获得遗传学，分子生物学和现代显微镜方法的经验，以研究细菌细胞生物学的问题。 对F.此外，johnsoniae亦会纳入多个本科生实验课程。从过去的经验来看，学生参与真实的研究，而研究结果事先并不知道（即使是教师也不知道），这会激发科学的好奇心，并以一种“食谱”实验室练习所不能的方式欣赏科学方法。为F. johnsoniae和拟杆菌门内相关细菌的研究结果将提供给科学界，研究结果将由学生在国家和国际会议上报告，并在主要期刊上发表。