Molecular Mechanisms of Organelle Assembly by the Bacterial Actin-Like Protein, M

描述（由申请人提供）：在本提案中，我们的目标是定义膜结合细胞器在趋磁细菌中组织的分子机制。这些微生物能够利用地球的磁场作为指导，在称为磁小体的专门细胞器的帮助下简化它们对低氧环境的搜索。磁小体是细胞内膜的小脂质双层内陷，其中产生纳米尺寸的铁基磁性颗粒。单个磁小体排列成一条链，使细菌与磁场的相互作用最大化。包括我们在内的许多小组的工作已经确定了大量基因在构建磁小体细胞器中具有特定的作用。其中一个因子MamK是一种细菌肌动蛋白样蛋白，位于磁小体链形成的中心。在没有MamK的情况下，磁小体不能形成连贯的链，而是被许多间隙分开。细菌肌动蛋白广泛存在于细菌中，形成许多具有不同功能的家族。例如，MreB参与指导细胞壁的合成，而ParM和其他肌动蛋白有助于分离天然存在的质粒。除了MreB和ParM之外，对绝大多数细菌肌动蛋白样蛋白的功能和行为知之甚少。MamK是其中一个家族的创始成员，其代表大多在MB中找到。在过去四年的资助下，我们开发了一种体外系统来研究MamK的聚合动力学，并通过合作获得了其细丝的高分辨率电子显微镜结构。此外，我们确定了两种蛋白质，MamJ和LimJ是负责MamK丝在体内的动态营业额。使用定向诱变策略，我们还确定了MamK的内在特征，有助于其功能，定位，捆绑和动力学。最后，我们使用更广泛的遗传策略来定义超过20个基因在磁小体形成和磁性颗粒生物矿化中的功能。在这项提案中，我们将利用我们的独家专业知识和基础设施，更详细地定义MamK功能和磁小体形成的机制。首先，我们将询问MamK的生化和结构特征是否在其家族的不同成员中表现出来，其中一些存在于非趋磁细菌和古细菌中。其次，我们将定义和表征MamK的调节器和相互作用。第三，我们将开发工具来询问MamK是否参与了链的建立、维护或隔离。最后，我们将开发工具来成像和理解磁小体膜生物发生的最早步骤。这些实验有望揭示细菌细胞器的细胞生物学和细菌肌动蛋白的进化和功能多样性的机制。

DESCRIPTION (provided by applicant): In this proposal we aim to define the molecular mechanisms by which membrane-bound organelles are organized in magnetotactic bacteria. These microorganisms are capable of using the earth's magnetic field as a guide to simplify their search for low oxygen environments with the help of a specialized organelle termed the magnetosome. Magnetosomes are small lipid-bilayer invagination of the inner cell membrane within which nanometer-sized iron-based magnetic particles are produced. Individual magnetosomes are arranged into a chain that maximizes the interaction of the bacterium with magnetic fields. The work of many groups, including ours, has pinpointed a large number of genes as having specific roles in building the magnetosome organelle. One of these factors, MamK, is a bacterial actin-like protein that is at the center of magnetosome chain formation. In the absence of MamK, magnetosomes fail to form coherent chains and are instead separated by numerous gaps. Bacterial actins are widespread amongst the Bacteria and form numerous families with distinct functions. For instance, MreB is involved in directing the synthesis of the cell wall whereas ParM and other actins help in segregation of naturally occurring plasmids. Outside of MreB and ParM, little is known regarding the function and behavior of the vast majority of the bacterial actin-like proteins. MamK is the founding member of one of these families and its representatives are mostly found within the MB. With the support of this grant over the last four years, we have developed an in vitro system to study the polymerization kinetics of MamK and, through collaborations, obtained a high-resolution electron microscopic structure of its filaments. Furthermore, we identified two proteins, MamJ and LimJ that are responsible for the dynamic turnover of MamK filaments in vivo. Using a directed mutagenesis strategy, we have also identified intrinsic features of MamK that contribute to its function, localization, bundling and dynamics. Finally, we have used broader genetic strategies to define the functions of over 20 genes in the formation of magnetosomes and biomineralization of magnetic particles. In this proposal, we have will build on our exclusive expertise and infrastructure to define the mechanisms of MamK function and magnetosome formation in more detail. First, we will ask if the biochemical and structural features of MamK are represented in the diverse members of its family, some of which exist in non-magnetotactic bacteria as well as the archaea. Second, we will define and characterize regulators and interactors of MamK. Third, we will develop tools to ask if MamK is involved in chain establishment, maintenance or segregation. Finally, we will develop tools to image and understand the earliest steps of magnetosome membrane biogenesis. These experiments promise to shed mechanistic light on the cell biology of bacterial organelles and the evolution and functional diversity of bacterial actins.