A New Interdisciplinary Frontier: Merging Molecular Biology with Isotopic Geochemistry

最近受到重视的微生物多样性范围暗示了环境中隐藏的巨大新陈代谢复杂性。这些过程支配着碳和能量在自然系统中的转移。然而，在所有已知的原核生物中，只有不到1%可以在纯培养中生长。需要新的、不依赖于培养的方法来研究不可培养原核生物的生态作用和代谢途径。这个项目将证明，核酸的碳同位素分析，特别是核糖体RNA(RRNA)，可以用于研究微生物的新陈代谢。任何样品中的天然rRNA都是分子生物学对“生物标记物”概念的类比。实现这一重要步骤的关键是最近发展出一种可行的分析方法，用于RNA分离和同位素分析。使用磁珠捕获法从总RNA的混合物中分离出完整的、物种特异性的rRNA。纯净的物质被转移到连接到同位素比质谱仪的移动导线接口。这是一项真正的跨学科成就，因为它允许分析纳克大小的rRNA样本的13C/12C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。科学目标这项初步研究将集中在地球生物学最近感兴趣的话题上。(I)海洋浮游考古动物碳同化的代谢途径是什么？这种生物使用3-羟基丙酸(3-HP)途径的假设正确吗？这种同位素分馏在自然界中的大小与实验室培养中的大小相同吗？如果不是，关于3-HP途径在自然群落中的作用，我们可能了解到什么？(2)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)对深层生物圈总生产力的贡献有多大？是否有可能区分AMO所涉及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P.I.，还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。这项工作还支持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从这项工作中产生；一些抽样将由本科生执行，作为必修学术课程的实地部分。

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.