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

该奖项由2009年美国复苏和再投资法案(公法第111-5条)资助。微生物是地球上最古老的生物，跨度约35亿年，它们在塑造地球--S土壤、海洋和大气--中的重要性早已被人们所接受。趋磁细菌生物合成磁铁矿(Fe3O4)是一个有趣的例子，因为它在催化、电子学、纳米技术和生物医学等领域的应用非常重要，它涉及到地球上生命的起源和进化的哲学含义，以及它参与自然环境中铁、氮、硫和碳的生物地球化学循环的潜力。此外，微生物对铁的生物地球化学循环(例如趋磁性细菌将铁积累和转化为Fe3O4)具有特别重要的意义，因为铁是表层和次表层环境中普遍存在的非常活跃的成分，因此影响区域和全球范围的气候和生态现象。此外，尽管铁无处不在，而且因为它的反应性，但它往往是生物体生长的限制因素，例如，在世界的一些地区--S海洋。从矿化、生物控制成核和定向生长的角度来看，纳米矿物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多年前就被发现了，但这些微生物中Fe3O4生物矿化的机制仍然不清楚。本研究的目的是利用原子力(单分子抗体识别力显微镜)和荧光显微镜/光谱学(时间分辨荧光各向异性和荧光共振能量转移)的单分子技术来确定趋磁细菌中纳米磁铁矿晶体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(S)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结构以及相对于新生的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性蛋白质复合体。拟议的活性会产生更广泛的影响吗？了解细菌指导合成Fe3O4纳米粒子的分子机制是生物启发材料合成的重要范式，将为包括多细胞生物体在内的其他有机体使用的受控晶体合成策略提供巨大的洞察力。通过了解趋磁细菌中Fe3O4的生物矿化过程，我们可能会了解如何确定环境中的Fe3O4颗粒是否为生物起源，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的证据。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性质的详细了解，而且由于微生物中的矿化过程本质上受纳米结构(例如蛋白质)的控制，这些知识将成为生物控制方法的基础，以合成定制的无机纳米结构，用于各种技术的应用。最后，研究人员相信，该项目开发的新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个PI和两名博士生(每个实验室一名)之间的新合作，这两名研究生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家会议以及每所大学的当地研讨会上展示他们的发现。第一位PI是职业生涯早期的教员，他曾帮助开创开发成像技术的先河，在分子水平上研究地球生物过程；第二位PI是资深教员，是世界知名的磁铁矿生物矿化权威，在该领域发表了150多篇论文。这项提案还将资助一名在Lead-Pi？S实验室工作的女性博士生。这项研究的结果将被纳入目前由私人投资促进机构教授和开发的本科生和研究生课程。此外，这项提案还将通过亲身实践演示和演讲，支持对小学、初中和高中生进行纳米地质科学和生物地球化学等新兴但往往被忽视的领域的教育。这些努力将鼓励大学预科学生从事生物地球化学职业和/或成为负责任的环境乘务员。

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.