Molecular Mechanisms of Organelle Assembly by the Prokaryotic Actin Homolog MamK

描述(由申请人提供)：在理解细菌细胞超微结构组织的分子机制方面的进展模糊了用于区分真核细胞和原核细胞的传统界限。其中最重要的是证明了原核生物含有真核生物微管蛋白、肌动蛋白和中间丝的功能和结构同源物。这些细胞骨架蛋白对多种原核生物的细胞分裂、细胞形状维持和染色体分离是必不可少的，有望成为发现新的抗菌剂的靶标。最近，我们发现趋磁细菌的磁小体细胞器的亚细胞组织需要细菌肌动蛋白同源物MamK。这些膜状细胞器被生物体用来产生纳米大小的磁性晶体，在细胞内以链的形式组织起来，并被独特的细胞骨架细丝网络包围。在没有MamK的情况下，这种细丝网络无法形成，磁小体分散在整个细胞中。系统发育分析表明，MamK是在趋磁细菌外发现的，它形成了细菌肌动蛋白样蛋白的一个独特分支。由于它不是生存所必需的，并且在细胞内有明显的定位，因此它可以作为一个易于处理的系统来深入分析细菌肌动蛋白样蛋白。我们对这项提议有以下目标。1)确定MamK的体外生化和生物物理性质。2)通过整体诱变策略确定MamK表面的功能结构域，然后进行一系列体内分析。3)鉴定和确定MamK相互作用蛋白的功能。4)通过对磁小体岛的遗传分析，确定参与MamK组装和磁小体膜形成的基因。磁小体岛是一个包含大多数已知磁小体基因的大基因组区域。除了它们与细菌肌动蛋白同源物的一般相关性外，这些发现还将为细菌中细胞器的形成过程提供洞察力。

DESCRIPTION (provided by applicant): Advances in understanding the molecular mechanisms underlying the ultrastructural organization of bacterial cells have blurred the traditional boundaries used to distinguish eukaryotic cells from their prokaryotic counterparts. Most significant amongst these has been the demonstration that prokaryotic organisms contain functional and structural homologs of eukaryotic tubulin, actin and intermediate filaments. These cytoskeletal proteins are essential for cell division, cell shape maintenance and chromosome segregation in a variety of prokaryotic species and hold promise as targets for the discovery of new antimicrobial agents. Recently, we showed the bacterial actin homolog, MamK, is required for the subcellular organization of the magnetosome organelles of magnetotactic bacteria. These membranous organelles, used by the organism to produce nanometer-sized magnetic crystals, are organized as chains within the cell and surrounded by a distinct cytoskeletal network of filaments. In the absence of MamK this network of filaments fails to form and the magnetosomes are dispersed throughout the cell. Phylogenetic analysis shows that MamK is found outside of the magnetotactic bacteria and that it forms a distinct branch of the bacterial actin like proteins. Since it is not essential for survival and has a distinct localization within the cell it may serve as a tractable system for in-depth analysis of bacterial actin-like proteins in general. We have the following goals for this proposal. 1) Define the biochemical and biophysical properties of MamK in vitro. 2) Identify functional domains on the surface of MamK through a global mutagenesis strategy followed by a series of in vivo analyses. 3) Identify and define the function of MamK-interacting proteins. 4) Determine the genes involved in MamK assembly and magnetosome membrane formation through a genetic analysis of the magnetosome island, a large genomic region containing most of the known magnetosome genes. In addition to their relevance to bacterial actin homologs in general these findings will provide insights into the process of organelle formation in bacteria.