Racemization dating of subsurface microorganisms

该项目旨在回答关于地下微生物群落代谢和生长速度的问题。微生物群落负责转化深埋在海洋和陆地沉积物中的有机物，以及通过热液过程和岩石/水相互作用（如蛇纹石化）在深部产生的有机物。这些有机物质要么转化为二氧化碳和甲烷，它们通常会返回地表并进入大气，要么转化为更多的微生物生物质。高度生物降解的石油是这些微生物转化的残余物。 地下微生物群落还调节地下水中铁、铀、砷和其他有毒金属和有机污染物的浓度。它们引起矿物相的成岩变化，如长石颗粒转变为粘土，并改变含水层的孔隙度和渗透率。然而，所有这些地球化学过程发生在我们脚下很远的地方，其速度受到很大的限制。该项目旨在开发一种新的方法，通过确定微生物本身的平均年龄，更准确地量化地下微生物生物地球化学过程的原位速率。如果微生物生长迅速，那么地球化学循环一定也同样快，但如果它们非常古老，那么地球化学过程的发生速度非常慢。在过去的20年里，碳转化的地球化学模型已被用来推断这些群落的原位代谢率和合成代谢率。根据所作的假设和所用的测量，对任何地点/样本的这些速率的估计值相差三到四个数量级。研究人员和他的同事最近提出，天冬氨酸（Asp）的右手和左手构型（D/L）的分析提供了对地下微生物合成代谢速率的直接约束。这些结果表明，嗜热微生物的蛋白质周转时间不到一年，支持这种营业额所需的地球化学速率比以前猜测的地球化学模型要大得多。随着测量方案和测试的进一步改进，Asp D/L分析可以提供比以前方法更准确的原位合成代谢率约束。当该速率与元蛋白质组学分析相结合时，还可以对最丰富的代谢途径的速率进行限制，从而对来自单个样品分析的生物化学转化进行限制。这一估计完全独立于过去使用的地球化学模型或同位素分析。为了实现这一目标，将在2年的时间内对目前存在完整基因组的孢子形成和非孢子形成嗜热菌和超嗜热菌进行饥饿实验。然后将测定作为温度函数的天冬氨酸外消旋化速率。将使用蛋白质组学绘制主要代谢途径，以确定ATP产生和消耗的速率。然后将其与作为温度的函数的测量的呼吸速率进行比较。如果外消旋化速率和异天冬氨酸形成速率代表主要的衰弱过程，则根据天冬氨酸的D/L和代谢途径分析得出的呼吸速率估计值应与测量速率一致。这些实验还将揭示天冬氨酸的D/L在什么程度上如此之高，以至于微生物真正死亡，再也不会复活。

This project aims to answer the question regarding how quickly subsurface microbial communities metabolize and grow. Microbial communities are responsible for the transformation of organic matter that is deeply buried in marine and terrestrial sediments and of organic matter generated at depth by hydrothermal processes and rock/water interactions, such as serpentinization. This organic matter is either converted to carbon dioxide and methane, which often returns to the surface and enters the atmosphere, or into more microbial biomass. Heavily biodegraded petroleum is the residue of these microbial conversions. Subsurface microbial communities also mediate the concentrations of iron, uranium, arsenic and other toxic metals and organic contaminants in groundwater. They cause diagenetic alteration of mineral phases, such as the transformation of feldspar grains into clay and change the porosity and permeability of aquifers. The rate at which all of this biogeochemical processing occurs far beneath our feet, however, is poorly constrained. This project seeks to develop a new method of more accurately quantifying the in situ rates of subsurface microbial biogeochemical processes by determining the average age the microorganisms themselves. If the microorganisms are growing quickly then the biogeochemical cycling must be equally fast, but if they are very old, then the biogeochemical processes are occurring at very slow rates. For the past 20 years geochemical models for carbon transformation have been used to deduce in situ metabolic rates and anabolic rates of these communities. Depending upon the assumptions made and measurements used, estimates of these rates vary by three to four orders of magnitude for any site/sample. The investigator and his colleagues recently proposed that the analyses of the right and left-hand configuration (D/L) of aspartic acid (Asp) provides a direct constraint on the anabolic rate of subsurface microorganisms. These results suggest that the protein turnover times for thermophilic microorganisms are less than one year and that the biogeochemical rates required to support this turnover are much greater than previously guessed from geochemical models. With further refinements in measurement protocols and testing, the Asp D/L analyses could provide a far more accurate constraint on the in situ anabolic rate than previous approaches. When this rate is combined with metaproteomic analyses, then constraints can also be placed on the rates of the most abundant metabolic pathways and thus biogeochemical transformations from analysis of a single sample. This estimate is completely independent of geochemical models or isotopic assays that have been used in the past. To realize this goal starvation experiments will be performed over a period of 2 years on spore-forming and non-spore forming thermophiles and hyperthermophiles for which complete genomes currently exist. The rate of racemization of aspartic acid as a function of temperature will then be determined. The principal metabolic pathways will be mapped using proteomics to determine the rate of ATP production and consumption. This will then be compared to the measured rates of respiration as a function of temperature. If the racemization rate and rate of isoaspartate formation represent the major debilitating process, then estimates of the respiration rate derived from the D/L of Asp and the metabolic pathway analysis should coincide with the measured rates. These experiments will also reveal at what point the D/L of aspartic acid is so high that a microorganism truly dies, never to come back to life.