Novel Motility and Protein Secretion Machinery of Flavobacterium Johnsoniae

摘要智力优势：强生黄杆菌的细胞通过一种被称为滑动运动的过程在表面爬行。这些细胞没有研究得很好的运动细胞器，如鞭毛或IV型菌毛。取而代之的是，他们依赖于一种新型的运动机器，这种机器由类杆菌门特有的蛋白质组成。目前已鉴定出24种与运动有关的蛋白质。这些蛋白质包括1)GLD蛋白质，它们是滑行的“马达”的组成部分；2)移动的细胞表面粘附素(SprB、REMA等)，它们似乎是由“马达”推动的，其功能可能有点像坦克踏板；以及3)新型蛋白质分泌系统(PorSS)的组成部分，这是将SprB组装到细胞表面所必需的。电子显微镜观察表明，SprB可能形成了从细胞延伸出来的长丝。SprB和REMA允许细胞在不同的表面上移动，它们具有凝集素样糖结合域，可能有助于与这些表面的结合。参与多糖合成和输出的基因突变的细胞也会导致运动性缺陷。这些结果提出了一种滑动模型，在该模型中，滑动的“马达”推动粘附丝沿着细胞表面移动。细胞表达不同的粘附素(SprB、REMA等)以允许在不同的表面上运动，胞外多糖可能通过包裹表面并与粘附素相互作用来增强运动性。遗传学、细胞生物学、生化和电子显微镜方法将被用来解决与运动机械的细胞表面组件相关的3个具体目标。具体目的1.表征参与滑行的移动细胞表面粘附素。针对Rema的抗体将被用来检验Rema是一种可移动的细胞表面蛋白的假设，并确定是否需要PorSS来分泌Rema。荧光标记的表位标记的REMA也将用于可视化细胞运动过程中发生的动态变化。将分析可溶性糖对REMA功能的影响，以确定凝集素结构域的结合特异性。将产生重组版本的Rema来确定蛋白质各个结构域的功能，并将使用冷冻电子显微镜断层扫描来验证Rema和SprB是不同细胞表面细丝成分的假设。具体目标2.确定细胞外多糖在滑动中的作用。Wza和Wzc是与滑行相关的胞外多糖分泌系统的组成部分。胞外多糖将被分离和鉴定。有wza和wzc突变的细胞将被分析，以检验他们缺乏胞外多糖的假设。纯化的多糖将被用来确定它们是否与Rema的凝集素结构域相互作用，细胞外互补将决定外源提供的多糖是否克服了wza和wzc突变体的运动缺陷。具体目的3.表征参与细胞-表面粘附素组装的蛋白质分泌系统。遗传方法将被用来识别参与蛋白质分泌的PorSS的其他成分，并确定针对该系统的分泌蛋白质的特征。将研究蛋白质之间的相互作用，以解决与POSSs结构和功能相关的问题。广泛的影响：本科生、研究生和博士后科学家将进行研究。学生将获得遗传学、分子生物学以及现代光学和电子显微镜方法来研究细菌细胞生物学问题的经验。这些项目非常适合让学生接触到广泛的技术。学生和博士后科学家将接受科学写作和演讲技巧方面的广泛培训。对强生镰刀菌的研究将被纳入几门本科实验室课程。本科生将主要负责构建和表征POSS潜在组成部分的突变。研究结果将由学生在全国会议上报告，并发表在领先的期刊上，为约翰尼亚氏杆菌和相关细菌开发的基因工具将向科学界提供。在拟杆菌门的许多成员中都发现了类似的滑动运动和蛋白质分泌机制，包括对环境和农业具有重要意义的细菌。其中一些细菌是影响水产养殖系统的重要鱼类病原体，另一些细菌具有消化纤维素的新机制，可能有助于将植物材料转化为生物燃料。因此，更好地了解约翰氏杆菌的运动性和蛋白质分泌可能会有实际应用。

AbstractIntellectual merit:Cells of Flavobacterium johnsoniae crawl over surfaces by a process known as gliding motility. These cells do not have well-studied motility organelles such as flagella or Type IV pili. Instead they rely on a novel motility machine composed of proteins that are unique to the phylum Bacteroidetes. Twenty-four proteins involved in motility have been identified. These include 1) Gld proteins that are components of the gliding 'motor', 2) mobile cell surface adhesins (SprB, RemA, and others) that appear to be propelled by the 'motor' and may function somewhat like a tank tread, and 3) components of a novel protein secretion system (PorSS) that is required for assembly of SprB on the cell surface. Electron microscopic observations suggest that SprB may form long filaments extending from the cell. SprB and RemA allow movement of cells on different surfaces, and they have lectin-like carbohydrate-binding domains that may facilitate binding to these surfaces. Cells with mutations in genes involved in polysaccharide synthesis and export also result in motility defects. These results suggest a model for gliding in which the gliding 'motors' propel adhesive filaments along the cell surface. Cells express different adhesins (SprB, RemA and others) to allow movement on different surfaces, and exopolysaccharides might enhance motility by coating the surfaces and interacting with the adhesins. Genetic, cell biology, biochemical, and electron microscopic approaches will be used to address 3 specific aims related to the cell-surface components of the motility machinery. Specific Aim 1. Characterize the mobile cell-surface adhesins involved in gliding. Antibodies against RemA will be used to test the hypothesis that RemA is a mobile cell-surface protein and to determine if the PorSS is required for secretion of RemA. Fluorescently labeled epitope-tagged RemA will also be used to visualize dynamic changes that occur during cell movement. The effect of soluble sugars on RemA function will be analyzed to determine the binding specificity of the lectin domain. Recombinant versions of RemA will be generated to determine the functions of individual domains of the protein, and cryo-electron microscopy tomography will be used to test the hypothesis that RemA and SprB are components of different cell-surface filaments.Specific Aim 2. Determine the role of extracellular polysaccharides in gliding. Wza and Wzc are components of the putative exopolysaccharide secretion system linked to gliding. Exopolysaccharides will be isolated and characterized. Cells with mutations in wza and wzc will be analyzed to test the hypothesis that they are deficient in exopolysaccharides. Purified polysaccharides will be used to determine if they interact with the lectin domain of RemA and extracellular complementation will determine if exogenously supplied polysaccharide overcomes the motility defects of wza and wzc mutants. Specific Aim 3. Characterize the protein secretion system involved in assembly of the cell-surface adhesins. Genetic approaches will be used to identify additional components of the PorSS involved in protein secretion and to determine the features of secreted proteins that target them to this system. Protein-protein interactions will be studied to address questions related to structure and function of the PorSS.Broader Impacts:Undergraduate students, graduate students and postdoctoral scientists will conduct the research. Students will gain experience in genetics, molecular biology, and modern light and electron microscopic approaches to study questions of bacterial cell biology. The projects are well suited to expose students to a broad array of techniques. Students and postdoctoral scientists will receive extensive training in scientific writing and in presentation techniques. Research on F. johnsoniae will be incorporated into several undergraduate laboratory courses. Undergraduate students will have primary responsibility for constructing and characterizing mutations in potential components of the PorSS. Research results will be reported by students at national meetings and published in leading journals, and genetic tools developed for F. johnsoniae and related bacteria will be made available to the scientific community. Similar gliding motility and protein secretion machines are found in many members of the phylum Bacteroidetes, including bacteria of environmental and agricultural importance. Some of these bacteria are important pathogens of fish that impact aquaculture systems, and others have novel mechanisms for digesting cellulose and may aid conversion of plant material into biofuels. An improved understanding of F. johnsoniae motility and protein secretion may thus have practical applications.