A New Interdisciplinary Frontier: Merging Molecular Biology with Isotopic Geochemistry

最近发现的微生物多样性范围暗示了环境中隐藏的巨大代谢复杂性。 这些过程控制着自然系统中碳和能量的转移。 然而，在所有已知的原核生物中，只有不到百分之一可以在纯培养中生长。 需要新的，培养独立的方法来调查的生态作用和代谢途径的不可培养的原核生物。 该项目将证明核酸的碳同位素分析，特别是核糖体RNA（rRNA），可用于研究微生物代谢。 任何样品中的天然rRNA都是分子生物学中类似于地球化学中“生物标志物”概念的物质。 实现这一重要步骤的关键是最近开发了一种用于RNA分离和同位素分析的可行分析方法。 使用磁珠捕获方法从总RNA的混合物中分离完整的物种特异性rRNA。纯物质被转移到连接到同位素比质谱仪的移动线接口。 这代表了一个真正的跨学科成就，因为它允许分析纳克大小的rRNA样品的13 C/12 C比率。 从任何天然样品中同时获得同位素和系统发育信息是可能的。 科学目标这个初步研究将集中在地球生物学最近感兴趣的主题。 (i)海洋浮游泉古菌碳同化的代谢途径是什么？ 这种微生物使用3-羟基丙酸（3-HP）途径的假设是否正确？ 这种同位素分馏的程度在性质上与实验室培养中的相同吗？ 如果不是，我们可以从3-HP途径在自然群落中的作用中学到什么？(ii)我们能否确定沉积和地热环境中甲烷的主要途径？ 厌氧甲烷氧化（AMO）在多大程度上有助于深层生物圈的总生产力？ 是否有可能区分AMO中不同微生物类群之间的代谢差异？该项目促进研究机构之间以及生物学家和地球科学家之间的合作。 除了私家侦探有五个人代表三个研究实验室，他们也将参与该项目的某些方面。 这项工作也支持NSF的使命，以促进研究生和本科教育的发展。 一个研究生博士。论文将直接从这项工作中产生;和一些抽样将由本科生作为一个必要的学术课程的领域组成部分进行。

The recently appreciated scope of microbial diversity hints at vast hidden metabolic complexity in the environment. These processes govern the transfer of carbon and energy in natural systems. However, less than one percent of all known prokaryotes can be grown in pure culture. New, culture-independent methods are required to investigate the ecological roles and metabolic pathways of uncultivable prokaryotes. This project will demonstrate that carbon isotopic analysis of nucleic acids, specifically ribosomal RNA (rRNA), can be used to study microbial metabolism. The native rRNA in any sample is molecular biology's analogue to the "biomarker" concept of geochemistry. The key to realizing this important step is the recent development of a viable analytical method for RNA separation and isotopic analysis. Intact, species-specific rRNA is separated from mixtures of total RNA using magnetic bead capture methods. The pure material is transferred to a moving-wire interface attached to an isotope-ratio mass spectrometer. This represents a truly interdisciplinary achievement, as it permits the analysis of 13C/12C ratios of nanogram-sized samples of rRNA. It is possible to obtain both isotopic and phylogenetic information, simultaneously, from any natural sample. Scientific Goals This preliminary study will focus on topics of recent interest in geobiology. (i) What is the metabolic pathway of carbon assimilation in marine planktonic crenarchaeota? Is the hypothesis that this organism uses the 3-hydroxypropionate (3-HP) pathway correct? Is the magnitude of this isotopic fractionation the same in nature as it is in laboratory cultures? If not, what might be learned about the role of the 3-HP pathway in natural communities? (ii) Can we identify the major pathway to CH4 in sedimentary and geothermal environments? To what extent does anaerobic methane oxidation (AMO) contribute to total deep biosphere productivity? Is it possible to distinguish metabolic differences between different microbial taxa involved in AMO? The project fosters collaboration among research institutions and between biologists and geoscientists. In addition to the P.I., there are five individuals representing three research laboratories who also will participate in some aspect of the project. This work also supports the mission of NSF to foster educational development at the graduate and undergraduate levels. A graduate student Ph.D. thesis will result directly from this work; and some of the sampling will be performed by undergraduate students as the field component of a required academic course.