IEP: Deconstructing bacterial community biomass stoichiometry one cell at a time

在过去的十年里，我们揭示生物世界复杂性的能力突飞猛进，这主要归功于我们能够读取实验室无法培养的微生物的基因序列。这些过多的信息揭示了环境微生物的多样性，超出了我们最疯狂的估计。生活在几乎所有环境中的微生物负责维持对地球上所有生命至关重要的生态系统服务。然而，即使在对微生物生物多样性的理解方面取得了进展，我们仍然几乎没有能力将特定微生物的存在与它正在执行的任务联系起来。这项研究通过开发和应用新的工具来解决这一差距，以了解环境中活细胞中含有哪些分子（蛋白质，核酸，脂质）以及某些元素（例如氮和磷）的数量，而无需在实验室中单独培养它们。更重要的是，拟议的研究提供了一个机会，将遗传生物多样性的信息与在环境中生长的细胞的分子和元素组成联系起来（即没有培养）。这些方法将使我们能够开始推断微生物如何在不同条件下分配限制性资源，以及它们如何改变限制性营养素对其他生物的可用性。从这一推论将提高我们对地球上生命的基本限制和生物多样性在维持所有生物体所必需的生态系统过程中的作用的理解。PI建议将现有的生态理论（生态化学计量学）应用于自然微生物群落，使用新的分子和单细胞技术来解决环境微生物学中的紧迫问题。他建议将拉曼显微光谱分析（其允许分析单细胞的大分子含量）与X射线微量分析（其允许分析单细胞的元素组成）结合原位杂交（例如FISH或EL-FISH，其允许在荧光（拉曼MS）和透射电子（XRMA）显微镜中可视化的细胞的系统发育鉴定）。每种方法都是为环境微生物学而建立的，然而，将分子微生物学与单细胞方法相结合以扩展生态学理论的应用，为理解对重要生态系统参数（微生物群落化学计量）的控制和约束提供了一种独特而新颖的方法。虽然微生物对所有生态系统的功能都非常重要，但我们理解它们如何影响最基本的生态系统功能（例如二氧化碳释放）的能力受到自然微生物群落固有的遗传和代谢多样性的异常复杂性的限制。对直接从环境中提取的微生物基因进行测序极大地增加了我们对环境中微生物多样性的广度和深度的理解。然而，关于一个群落的系统发育结构的信息往往没有伴随着关于该生物体的生理学或表型的任何额外信息。该项目将使用新的经验工具来跟踪自然微生物群落对营养添加物的反应，从大分子的相对丰度到整个群落的系统发育结构的变化。这项研究将推进目前对复杂微生物群落如何改变基本生态系统过程（即营养循环）的理解。虽然该项目的重点是水生浮游生物群落，但由于它植根于微生物生理学和生态学理论，因此其结果应适用于分析各种生态系统的微生物群落。

Over the last ten years our ability to uncover the complexity of the living world has increased in leaps and bounds largely due to our ability to read the genetic sequence of microorganisms that cannot be grown in the laboratory. This plethora of information has revealed a diversity of environmental microorganisms beyond our wildest estimates. Microbes that live in virtually all environments are responsible for maintaining the ecosystem services that are vital for all life on earth. However, even with advances in understanding of microbial biodiversity we still have very little ability to link the presence of a given microorganism to the task it is performing. This research addresses that gap by developing and applying new tools to understand which molecules (proteins, nucleic acids, lipids) and how much of certain elements (e.g. nitrogen and phosphorus) are contained within living cells in the environment without having to grow them separately in the laboratory. More importantly, the proposed research provides an opportunity to link the information on genetic biodiversity with molecular and element composition of cells growing in the environment (i.e. without culturing). These approaches will allow us to began to make inferences on how microorganisms partition limiting resources under different conditions and how they alter availability of limiting nutrients to other organisms. Inferences from this will improve our understanding of fundamental constraints of life on earth and the role of biodiversity in maintaining the ecosystem processes essential for all living organisms.The PI proposes to apply existing ecological theory (Ecological Stoichiometry) to natural microbial communities using novel molecular and single-cell techniques to address pressing questions in environmental microbiology. He has proposed to couple analyses of Raman microspectroscopy (which allows for analysis of macromolecular content of single cells) with xray microanalysis (which allows for analysis of elemental composition of single cells) with in situ hybridization (e.g. FISH or EL-FISH, which allows for phylogenetic identification of cells visualized in both eplifluorescence (Raman MS) and transmission electron (XRMA) microscopy). Each method is established for use in environmental microbiology however the synthesis of molecular microbiology with single cell approaches to extend the application of ecological theory provides a unique and novel approach to understanding the controls and constraints on an important ecosystem parameter (microbial community stoichiometry). While microorganisms are exceptionally important to the functioning of all ecosystems our ability to understand how they influences even the most basic ecosystem function (e.g. CO2 release) is limited by the exceptional complexity of the genetic and metabolic diversity inherent in natural microbial communities. Sequencing microbial genes directly extracted from the environment has dramatically increased our understanding of the breadth and depth of microbial diversity in the environment. However, information on phylogenetic structure of a community often comes unaccompanied with any additional information on that organism's physiology or phenotype. This project will use new empirical tools to track the response of natural microbial communities to nutrient additions from the relative abundance of macromolecules through changes in phylogenetic structure to the elemental composition of the whole community. The research will advance the current understanding of how complex microbial communities alter fundamental ecosystem processes (i.e. nutrient cycling). While this project focuses on aquatic planktonic communities, because it is rooted in microbial physiology and ecological theory the results should be applicable to analysis of microbial communities from a wide range of ecosystems.