An integrated approach for ecological, cultivational, ultrastructural, and genomic analysis of the uncultivated giant magnetotactic rod cand. "Magnetobacterium bavaricum"

趋磁性细菌(MTB)是一类高度多样化的水生原核生物，其游泳运动由复杂的细胞内磁性细胞器--磁小体控制，这些细胞器是由受控的生物矿化作用形成的。尽管它们在许多水生生态系统中具有重要的生态意义和丰度，但它们的生物学在很大程度上仍然不为人所知，因为大多数来自自然种群的结核杆菌抵制传统的隔离尝试。我们计划对未开垦的磁致伸缩棒棒进行表征。与所有其他已知的MTB不同的是，它与深分支的Nitrospira群有独特的从属关系，其复杂的细胞生物学和巨大的体积，以及它能够生物矿化多达1000个子弹状的磁铁矿晶体。在拟议的项目中，我们计划通过一套创新方法的综合方法来研究其生态学、生理学、细胞生物学和遗传学特征。通过微电极测量从两个淡水采样点，包括一个专门的中微体获得的地球化学特征将与细胞的丰度和空间分布相关联。巴伐利亚分枝杆菌的细胞将从环境微观世界中进行磁浓缩，通过各种方法进行彻底的超微结构分析，包括冷冻切片和FIB-研磨，然后是扫描电子显微镜和电子断层扫描。我们将建立对巴伐利亚分枝杆菌进行部分并最终完成基因组分析的先决条件。首先，从收集的细胞中分离出的基因组DNA将用于构建大插入Fosid文库，该文库将用于筛选已知磁小体岛基因簇的同源物。其次，通过显微操作挑选的细胞将用于全基因组扩增实验，并对扩增出的DNA进行焦磷酸测序。最后，来自生态和基因组特征的结果将被用来指导高通量栽培方法。

Magnetotactic bacteria (MTB) are a highly diverse group of aquatic procaryotes whose swimming motility is directed by complex intracellular magnetic organelles, the magnetosomes, which are formed by controlled biomineralization. Despite of their ecological importance and abundance in many aquatic ecosystems, their biology has remained largely unknown, since most MTB from natural populations have resisted conventional attempts of isolation. We plan to characterize the uncultivated magnetotactic rod cand. “Magnetobacterium bavaricum”, which is distinct from all other known MTB by its unique affiliation with the deep-branching Nitrospira group, its complex cell biology and large size, and its ability to biomineralize up to 1000 bullet-shaped magnetite crystals. In the proposed project we plan to investigate its ecology, physiology, cell biology and genetic characteristics by using an integrated approach comprising a set a of innovative methods. Geochemical characteristics obtained by microelectrode measurements from two freshwater sampling sites including a dedicated mesocosm will be correlated with the abundance and spatial distribution of cells. Cells of M.bavaricum will be magnetically enriched from environmental microcosms for thorough ultrastructural analysis by various methods including cryo-sectioning and FIB-milling followed by SEM and electron tomography. We will establish the prerequisites for partial and, ultimately, complete genome analysis of M. bavaricum. First, genomic DNA isolated from collected cells will be used for the construction of large-insert fosmid libraries that will be screened for homologs of known magnetosome island gene clusters. Second, cells selected by micromanipulation will be employed in whole genome amplification experiments, and amplified DNA will be subjected to pyrosequencing. Finally, results from ecological and genomic characterization will be used to guide a high-throughput cultivational approach.