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，其效率至少是二氧化碳的20倍。它目前的大气浓度约为1.8ppmv。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。生物圈中最重要的甲烷生物汇是旱地土壤，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)会被土壤中的一群特定微生物吸收。森林土壤中的细菌“旱地土壤α”分支(USCA分支)被认为是适应微量大气甲烷的甲烷营养细菌，在大气中甲烷的去除中起着至关重要的作用。我们从研究甲烷等具有气候重要性的气体的循环中知道，微生物的消耗是一个极其重要的过程，它受到气候变化的很大影响。全球变暖和土地利用变化将通过改变环境和土壤条件(如温度升高、水分变化、甲烷和二氧化碳浓度增加、森林砍伐和农业用氮肥)对森林土壤中的微生物产生重大影响。然而，由于USCA分支的细菌尚未在实验室中分离和培养，因此人们对这些微生物的身份、生理、生化和代谢能力以及它们如何响应环境变化(即它们的活动如何对环境变化做出反应)知之甚少。利用强大的基于分子生物学的技术，因此，我的目标是通过从土壤中分离这一分支的单个细胞，并查看细胞的DNA，即基因组，来研究USCA的身份和能力。大多数细菌和古生菌看起来很相似，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。我将使用基因组信息，其中包含微生物如何工作(微生物的新陈代谢蓝图)以及它在营养方面的需求，从大气浓度下具有高活性氧化甲烷的土壤中丰富和分离USCA分支的细菌。从USCA分支细菌中纯化负责高亲和力甲烷氧化活性的酶将为我们深入了解其甲烷氧化活性的调控提供重要的指导意义。此外，我将应用自己和合作者开发的工具来分析土壤样本和富集物的总DNA和RNA，分别称为元基因组学和元翻译组学。这将进一步使我们能够确定USCA细菌将如何对气温上升和土地利用变化做出反应。使用最先进的技术将使我能够识别森林土壤中大气甲烷氧化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，这些信息将被用于调查不同地理位置的USCA细菌的存在，并观察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强有力的机械数据，用于预测气候变化和土地利用对森林/旱地土壤中大气甲烷的全球汇强度的健康和功能的影响。

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.