Single cell genomics and characterisation of the atmospheric methane oxidizing clade USC alpha and their response to climate change

甲烷是一种非常有效的温室气体，其有效性至少是二氧化碳的20倍。它目前的大气浓度约为1.8 ppmv。大气甲烷的最大生物来源包括自然湿地、水稻农业、牲畜、垃圾填埋场、白蚁和海洋。在生物圈中，最重要的甲烷生物汇是高地土壤，特别是森林土壤，它显示出任何土壤生态系统中最大的甲烷氧化消耗能力。大气中的甲烷（浓度很低）被这些土壤中的一组特殊微生物吸收。森林土壤中的细菌“高地土壤簇α”分支（USCA分支），这是以前仅通过培养独立的分子生物学技术检测到的，被假定为代表甲烷营养菌适应于痕量水平的大气甲烷，在从大气中去除甲烷中发挥重要作用。通过研究甲烷等重要气候气体的循环，我们知道微生物的消耗是一个极其重要的过程，受气候变化的影响很大。全球气候变暖和土地利用变化将通过改变环境和土壤条件对森林土壤中的微生物产生重大影响（例如，温度升高、含水量变化、甲烷和二氧化碳浓度增加、森林砍伐和农业使用导致的氮肥）。然而，由于USCa进化枝的细菌尚未在实验室中分离和培养，关于这些微生物的身份、生理学、生物化学和代谢能力以及它们如何响应环境变化（即它们的活动如何响应环境变化而被调节）所知甚少。使用强大的基于分子生物学的技术，因此，我的目标是调查身份和能力的USCa通过分选单细胞的这个分支从土壤中，并期待在DNA的细胞，基因组。大多数细菌和古细菌看起来很相似，所以我们经常使用DNA和RNA序列来研究它们在自然界中的作用和活动。我将使用包含微生物如何工作（微生物的代谢蓝图）以及它在营养方面所需的基因组信息，从在大气浓度下氧化甲烷具有高活性的土壤中富集和分离USCa分支的细菌。从USCa分支的细菌中纯化负责高亲和力甲烷氧化活性的酶将使我们对其甲烷氧化活性的调节有重要的了解。此外，我将应用自己和合作者开发的工具来分析土壤样品和富集物的总DNA和RNA，分别称为宏基因组学和元转录组学。这将进一步使我们能够确定USCa细菌将如何对温度升高和土地利用变化作出反应。使用最先进的技术将使我能够识别森林土壤中的大气甲烷氧化USCa细菌，并确定它们的活动如何受到气候变化和土地利用变化（例如森林砍伐和农业实践变化）的控制和影响。此外，本发明还提供了一种方法，这些信息将用于调查不同地理位置的USCA细菌的存在，并研究不同环境对这些微生物进化的长期影响。该项目的结果将提供可靠的机制数据，用于预测气候变化和土地利用对森林/高地土壤中大气甲烷的健康和全球汇强度的影响。

Methane is a very potent greenhouse gas, at least 20 times more effective than carbon dioxide. It has a current atmospheric concentration of about 1.8 ppmv. The largest biogenic sources of atmospheric methane include natural wetlands, rice agriculture, livestock, landfills, termites and oceans. The most important biological sinks for methane in the biosphere are upland soils, especially forest soils which show the greatest methane oxidation consumption capability of any soil ecosystem. Methane from the atmosphere (ie at very low concentrations) is taken up by a specific group of microorganisms in those soils. The bacterial "upland soil cluster alpha" clade (USCa clade) in forest soils, which has been previously detected solely by cultivation-independent molecular biological techniques, is assumed to represent the methanotrophic bacteria adapted to the trace level of atmospheric methane which play an essential part in the removal of methane from the atmosphere. We know from studying the cycles of climatically important gases such as methane that microbial consumption is an extremely important process which is greatly influenced by climate change. Global warming and land use change will have a significant impact on the microorganisms in forest soil by changing the environmental and soil conditions (e.g. increased temperature, changing water content, increased methane and carbon dioxide concentration, deforestation and nitrogen fertilization due to agricultural use).However, since bacteria of the USCa clade have not be isolated and grown in culture in the laboratory, little is known about the about the identity, the physiology, biochemistry and metabolic capabilities of these microorganisms and how they respond to changes in the environment ie how their activities are regulated in response to environmental change.Using powerful molecular biology-based techniques, I therefore aim to investigate the identity and abilities of the USCa by sorting single cells of this clade from soil and looking at the DNA of the cells, the genome. Most bacteria and archaea look alike, so we frequently use DNA and RNA sequences to study their roles and activity in nature. I will use the genome information which contains how a microorganism works (the metabolic blueprint of the microorganism) and what it needs in terms of nutrients, to enrich and isolate bacteria of the USCa clade from soils that have high activities in oxidising methane at atmospheric concentrations. The purification of the enzyme responsible for the high-affinity methane oxidation activity from bacteria of the USCa clade will give us significant insight into the regulation of its methane oxidation activity. Additionally, I will apply tools developed by myself and collaborators to analyse total DNA and RNA of soil samples and enrichments, termed metagenomics and metatranscriptomics, respectively. This will further enable us to determine how the USCa bacteria will react to rising temperatures and changes in land use. The use of state-of-the-art techniques will allow me to identify the atmospheric methane oxidizing USCa bacteria in forest soil, and to determine how their activity is controlled and influenced by a changing climate and changes in land use (e.g. deforestation and changes in agricultural practice). In addition, this information will be used to survey for the presence of USCa bacteria in different geographical locations and to look at the long-term effect of different environments on the evolution of these microorganisms.The results of this project will provide robust mechanistic data with which to predict the impact of climate change and land use on health and functioning of the global sink strength for atmospheric methane in forest/upland soils.