A New Interdisciplinary Frontier: Merging Molecular Biology with Isotopic Geochemistry

最近人们对微生物多样性的认识暗示了环境中隐藏着巨大的代谢复杂性。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，只有不到百分之一能够在纯培养物中生长。需要新的、不依赖培养的方法来研究不可培养的原核生物的生态作用和代谢途径。该项目将证明核酸，特别是核糖体RNA （rRNA）的碳同位素分析可以用于研究微生物代谢。任何样本中的原生rRNA都是分子生物学中类似于地球化学中“生物标志物”概念的东西。实现这一重要步骤的关键是最近一种可行的RNA分离和同位素分析方法的发展。使用磁珠捕获方法从总RNA混合物中分离出完整的、物种特异性的rRNA。纯物质被转移到一个与同位素比质谱仪相连的移动线界面。这代表了一个真正的跨学科成就，因为它允许分析纳米克大小的rRNA样品的13C/12C比率。从任何自然样品中同时获得同位素和系统发育信息是可能的。科学目标本初步研究将集中在地球生物学最近感兴趣的主题上。(i)海洋浮游藻碳同化的代谢途径是什么？这种生物使用3-羟丙酸（3-HP）途径的假设是否正确？这种同位素分馏的幅度在自然界和在实验室培养中是相同的吗？如果没有，我们可以从3-HP通路在自然群落中的作用中学到什么？（ii）沉积和地热环境中CH4的主要生成途径能否确定？厌氧甲烷氧化（AMO）对深层生物圈总生产力的贡献有多大？是否有可能区分与AMO相关的不同微生物类群之间的代谢差异？该项目促进了研究机构之间以及生物学家和地球科学家之间的合作。除了私家侦探之外，还有代表三个研究实验室的五个人也将参与该项目的某些方面。这项工作也支持了国家科学基金会促进研究生和本科生教育发展的使命。研究生的博士论文将直接来自于这项工作；一些抽样将由本科生执行，作为必修学术课程的实地组成部分。

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.