Collaborative Research: Using Single-Molecule Force and Fluorescence Microscopy to Elucidate the Molecular Mechanism of Bioinspired Magnetite Synthesis in Magnetotactic Bacteria

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。微生物是地球上最古老的活着的居民，跨越了大约35亿年，它们在塑造地球方面的重要性？美国的土壤、海洋和大气早已被接受。趋磁细菌对磁铁矿（Fe3O4）的生物合成是一个有趣的例子，因为它在催化、电子、纳米技术和生物医学科学等应用中的重要性，它对地球上生命的起源和进化的哲学意义，以及它在自然环境中参与铁、氮、硫和碳的生物地球化学循环的潜力，引起了人们的极大兴趣。此外，微生物对铁的生物地球化学循环（例如，趋磁细菌将铁积累并转化为Fe3O4）尤为重要，因为铁是地表和地下环境中普遍存在且非常活跃的成分，因此影响区域和全球尺度的气候和生态现象。此外，尽管铁无处不在，而且由于它的反应性，它往往是生物生长的限制因素，例如，在世界上的某些地区？年代的海洋。从矿化的角度来看，生物控制成核和纳米矿物的定向生长是复杂分子系统中自组织的一个很好的例子。尽管趋磁细菌在30多年前被发现，但这些微生物中Fe3O4生物矿化的机制仍然未知。本研究的目的是利用原子力单分子技术（单分子抗体识别力显微镜）和荧光显微镜/光谱学技术（时间分辨荧光各向异性和荧光共振能量转移）来确定纳米磁铁矿晶体在致磁细菌体内生物矿化的分子机制。研究人员将确定参与生物矿化过程的单个蛋白质分子的功能，并确定它们如何控制晶体成核、生长和形态，检查细菌膜内蛋白质分子的组织，以及新生的Fe3O4纳米颗粒，确定这些分子中晶体成核和生长所需的氨基酸序列。并发现Fe3O4生物矿化所需的功能蛋白复合物。拟议的活动有何更广泛的影响？了解细菌指导Fe3O4纳米颗粒合成的分子机制代表了生物启发材料合成的一个重要范例，这将为其他生物（包括多细胞生物）使用的受控晶体合成策略提供巨大的见解。通过了解趋磁细菌中Fe3O4的生物矿化过程，我们可以了解如何确定环境中的Fe3O4颗粒是否具有生物源性，这反过来可能为使用环境中发现的Fe3O4晶体作为地球上过去生命的生物标志物提供可靠性证据。此外，由于技术进步往往依赖于对单晶、复合材料、界面和纳米晶体的材料特性的详细了解，并且由于微生物中的矿化过程本质上是由纳米级结构（例如蛋白质）控制的，因此这些知识将成为生物控制方法合成定制无机纳米结构的基础，用于各种技术的应用。最后，研究人员相信，该项目开发的新型成像技术将成为强大的工具，可用于其他地球生物学或生物系统的研究。拟议的研究将支持两名pi和两名博士研究生（每个实验室一名学生）之间的新合作，他们将在这项研究中发挥不可或缺的作用，并鼓励他们在国际和国内会议以及每所大学的本地研讨会上展示他们的发现。一位PI是一名早期职业教师，他帮助开发了成像技术，以研究分子水平上的地球生物学过程；另一位PI是一名高级教师，他在磁铁矿生物矿化方面是世界知名的权威，在该领域发表了150多篇论文。此提案亦将资助1名在Lead- PI工作的女博士生。s实验室。这项研究的结果将被整合到pi目前教授和正在开发的本科和研究生课程中。此外，该提案将支持通过实际演示和演示来教育小学、初中和高中年龄的学生，让他们了解纳米地球科学和生物地球化学这些新兴但经常被忽视的领域。这些努力旨在鼓励大学预科学生从事生物地球化学方面的职业和/或成为负责任的环境管理人员。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Microorganisms are the oldest living inhabitants of planet Earth, spanning some 3.5 billion years, and their importance in shaping the Earth?s soils, oceans, and atmosphere has long been accepted. The biosynthesis of magnetite (Fe3O4) by magnetotactic bacteria is an interesting example that has generated a great deal of interest because of its importance in applications such as catalysis, electronics, nanotechnology, and biomedical sciences, its philosophical implications concerning the origin and evolution of life on Earth, and its potential to participate in the biogeochemical cycling of iron, nitrogen, sulfur, and carbon in natural environments. Furthermore, the biogeochemical cycling of iron by microorganisms (e.g., the accumulation and conversion of iron into Fe3O4 by magnetotactic bacteria) is of particular importance because iron is a ubiquitous and very reactive constituent of surface and subsurface environments and, as a result, impacts regional and global scale climatic and ecological phenomena. In addition, despite its ubiquity and because of it reactivity, iron is often a limiting factor for growth of organisms, for example, in some parts of the world?s oceans. From the point of view of mineralization, biological control over nucleation and directed growth of nanominerals is an elegant example of self-organization in complex molecular systems.Despite the discovery of magnetotactic bacteria over 30 years ago, the mechanism for Fe3O4 biomineralization in these microorganisms remains unknown. The objective of this research is to use single-molecule techniques of atomic force (single-molecule antibody recognition force microscopy) and fluorescence microscopy/spectroscopy (time-resolved fluorescence anisotropy and fluorescence resonance energy transfer) to determine the molecular mechanism for the biomineralization of nanomagnetite crystals in magnetotactic bacteria. Investigators will identify the function(s) of the individual protein molecules involved in the biomineralization process and determine how they control crystal nucleation, growth and morphology, examine the organization of the protein molecules within a bacterial membrane and with respect to nascent Fe3O4 nanoparticles, identify the amino acid sequences within these molecules required for crystal nucleation and growth, and uncover functional protein complexes required for Fe3O4 biomineralization.The broader impacts resulting from the proposed activity ? Understanding the molecular mechanism by which bacteria direct the synthesis of Fe3O4 nanoparticles represents an important paradigm for bioinspired materials synthesis that would provide enormous insight into the strategies of controlled crystal synthesis used by other organisms, including multi-cellular organisms. By understanding the biomineralization process of Fe3O4 in magnetotactic bacteria, we might learn how to determine whether Fe3O4 grains in the environment are biogenic in origin, which, in turn, might provide evidence of reliability for the use of Fe3O4 crystals found in the environment to be used as biomarkers for past life on Earth. Furthermore, because technological progress often relies on a detailed understanding of the material properties of single crystals, composites, interfaces, and nanocrystals, and because the mineralization process in microorganisms is inherently controlled by nanoscale structures (e.g., proteins), this knowledge will become the basis for bio-controlled approaches to synthesize tailor-made inorganic nanostructures for applications across a diverse span of technologies. Finally, investigators believe that the novel imaging techniques developed as a result of this project will emerge as powerful tools that can be used for studies in other geobiological or biological systems.The proposed research will support a new collaboration between the two PIs and two Ph.D. graduate students (one student from each laboratory) who will play an integral role in this research and be encouraged to present their findings at international and national conferences and local seminars at each university. One PI is an early-career faculty member who has helped pioneer efforts to develop imaging techniques to study geobiological processes on a molecular level and the second PI is a senior faculty member who is a world-renowned authority in magnetite biomineralization and has authored over 150 publications in this field. This proposal will also fund 1 female PhD student who works in the Lead- PI?s laboratory. The results of this research will be integrated into the undergraduate and graduate courses currently taught and being developed by the PIs. Furthermore, this proposal will support efforts to educate elementary, middle-, and high school age students about the burgeoning yet often overlooked fields of nanogeoscience and biogeochemistry through hands-on demonstrations and presentations. These efforts will be geared to encourage pre-college students to pursue careers in biogeochemistry and/or become responsible stewardesses of the environment.