Regulatory Interplay between Nitrogen and Methane Metabolism: New Frontiers in Product Discovery, Physiology and Ecology of Methanotrophic Bacteria

单碳化合物的代谢是一种古老而广泛的生物功能，但只有甲基营养细菌专门利用单碳化合物作为能量和碳的来源。甲烷营养型（即以甲烷为食）甲烷营养型生物作为地球的甲烷生物汇是必不可少的，随着温室气体在这一全球快速变化时期的持续积累，理解这一点变得至关重要。通过对30多个甲烷化菌基因组序列的比较，揭示了氮代谢与碳代谢密切相关的丰富多样性。提出的研究的总体假设是，氮源和可用性可预测地控制甲烷营养细菌的碳代谢，而不考虑基因组变异性，使我实验室的学生能够：1)确定氮代谢如何调节碳转化为工业开发的增值终端产品；2)表征我们最近发现的甲烷依赖反硝化途径的酶、功能和调控；3)开发离散的分子标记，以确定甲烷依赖反硝化菌在不同环境中调节温室气体通量的存在、强度和相关性。我们基于实验室的方法利用我们管理和维护的大型基因组测序菌株收集以及一套分析工具来测量批量和连续培养中的细胞生长，实时微量气体测量，基因表达水平以及代谢物和蛋白质生产。我们在目标1中的目标是确定n源和可用性如何将碳转化为与工业相关的分子，如脂肪酸、类异戊二烯、n存储聚合物、c存储聚合物和有机酸。从氮和碳源的不同组合中培养的核酸、蛋白质和代谢物的生长动力学和分析将有助于发现用于工业开发的产品。目标2将确定硝酸盐中依赖甲烷的反硝化作用的分子调控，这是我实验室发现的一种新的生理学，它连接了单一生物体内两种强效温室气体（甲烷和一氧化二氮）和一种主要环境污染物（硝酸盐）的代谢。利用生理学、转录组学和蛋白质组学工具，我们的目标是充分表征这种活性的酶多样性和生理益处，并根据基因含量和调控特征预测特定菌株是否应该具有这种生理。目标3将揭示甲烷依赖反硝化的生态相关性，以及从奶牛瘤胃到融化的永久冻土等不同环境中执行甲烷依赖反硝化的微生物。反硝化氧化甲烷菌拥有系统发育上不同的基因，我们的目标是在以甲烷消耗为代价生产一氧化二氮的环境中发现这些基因。总之，拟议的研究充分利用我们的甲烷营养细菌基因组测序收集和分析能力，a)通过将工业废物转化为增值产品与加拿大能源部门对接，b)表征甲烷营养细菌的酶多样性和新生理功能。c)提供工具来评估依赖甲烷的反硝化作为碳和氮生物地球化学循环之间的新联系的生态意义。除了为加拿大和更广泛的科学界开发一个潜在的利润丰厚和生态重要的研究领域外，这项工作还为微生物学和生物技术的学生提供了独特的培训经验。

Metabolism of single-carbon compounds is an ancient and widespread biological function, yet only methylotrophic bacteria exclusively utilize single-carbon compounds as sources of energy and carbon. Methanotrophic (i.e. methane-eating) methylotrophs are essential as the Earth's biological methane sink, which is becoming critical to understand as greenhouse gases continue to accumulate during this period of rapid global change. Comparison of over 30 methanotroph genome sequences revealed a rich diversity of inventory for nitrogen metabolism that is intimately interwoven with carbon metabolism. The overarching hypothesis of the proposed research is that nitrogen source and availability predictably control carbon metabolism in methanotrophic bacteria, regardless of genomic variability, enabling students in my laboratory to: 1) determine how nitrogen metabolism regulates carbon transformations into value-added end products for industrial exploitation, 2) characterize enzymes, functionality, and regulation for our recently discovered pathway of methane-dependent denitrification, and 3) develop discrete molecular markers to determine the presence, strength, and relevance of methane-dependent denitrifiers in regulating greenhouse gas fluxes in diverse environments. Our laboratory-based approaches utilize a large genome-sequenced strain collection that we curate and maintain along with a suite of analytical tools to measure cell growth in batch and continuous culture, real-time trace gas measurements, gene expression levels, and metabolite and protein production. Our goal in Objective 1 is to determine how N-source and availability gate carbon into molecules of industrial relevance like fatty acids, isoprenoids, N-storage polymers, C-storage polymers, and organic acids. Growth kinetics and analysis of nucleic acids, proteins, and metabolites from cultures grown with variable combinations of nitrogen and carbon sources will enable discovery of products for industrial exploitation. Objective 2 will determine the molecular regulation of methane-dependent denitrification from nitrate, a novel physiology discovered in my laboratory, that connects the metabolism of two potent greenhouse gases (methane and nitrous oxide) and a major environmental pollutant (nitrate) within a single organism. Using physiological, transcriptomic and proteomic tools, our aim is to fully characterize the enzymatic diversity and physiological benefit of this activity and to predict, based on gene content and regulatory features, whether a particular strain should have this physiology. Objective 3 will reveal the ecological relevance of methane-dependent denitrification and the microorganisms that perform it in diverse environments from cow rumen to thawing permafrost. Denitrifying methanotrophs possess phylogenetically distinct genes that we aim to find in environments where nitrous oxide production at the expense of methane consumption has been noted. Together, the proposed research fully employs our genome-sequenced collection of methanotrophic bacteria and analytical capacity to a) interface with the energy sector in Canada by converting industrial waste products into value-added products, b) characterize the enzymatic diversity and function of a novel physiology in methanotrophic bacteria, and c) provide tools to assess the ecological significance of methane-dependent denitrification as a novel linkage between the carbon and nitrogen biogeochemical cycles. In addition to developing a potentially lucrative and ecologically important area of research for Canada and the broader scientific community, this work offers a unique training experience for students of microbiology and biotechnology.