Flavobacterium Gliding Motility: Role of Cell-Surface Components

智力优势：强johnsoniae黄杆菌的细胞通过一种被称为滑动运动的过程在表面上快速移动。近一个世纪以来，这种运动形式的机制一直是个谜。最近发现了12种滑翔所需的Gld（滑翔）蛋白。细胞表面蛋白被认为是滑翔机制的一部分，但令人惊讶的是，没有一个Gld蛋白暴露在细胞表面。用于鉴定Gld蛋白的遗传方法可能会错过具有重叠功能的冗余运动蛋白。对转座子诱导的部分运动缺陷突变体的筛选发现了新的突变，称为sprA， sprB， sprC和sprD，它们似乎编码了一些缺失的滑动机制表面成分。这些蛋白质可能是由Gld蛋白沿着细胞表面推进的粘附素。在野生型细胞上观察到细胞表面原纤维，但在sprB、sprC和sprD突变体缺失的细胞上却没有。这表明原纤维是由Spr蛋白组成的，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质；(2)可视化滑翔运动装置；(3)确定滑行机械的运动部件。分子实验将用于表征SprA、SprB、SprC和SprD，而新的遗传方法将用于识别运动机制的其他细胞表面成分。现代电子显微镜（EM）方法，包括低温电子显微镜断层扫描和免疫电子显微镜，将用于可视化运动装置。Spr蛋白的作用将通过确定特异性抗体对运动的影响，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颖而常见的细菌运动形式的机制，并将产生关于分子马达、转运体和粘附素的新信息。更广泛的影响：本科生和研究生将进行这项研究的大部分。这些学生将获得遗传学、分子生物学和现代微观方法研究细菌细胞生物学问题的经验。约翰逊氏杆菌的研究也将被纳入几个本科实验课程。从过去的经验来看，学生参与事先不知道结果的实际研究（即使是教师），会激发对科学的好奇心和对科学方法的欣赏，这是“食谱”式的实验室练习所无法做到的。为拟杆菌门内的johnsoniae和相关细菌开发的遗传工具将向科学界开放，研究结果将由学生在国内和国际会议上报告，并在主要期刊上发表。

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.