Role of methanobactin in methane oxidation rates in the presence of mineral copper

甲烷氧化细菌（甲烷氧化菌）是自然界降低大气甲烷水平的主要机制，甲烷是第二大温室气体。了解这些关键生物的丰度和分布，对于了解大自然对人为气体和其他释放物（如变暖导致的永久冻土带融化）增加的反应至关重要。不幸的是，尽管这些生物很重要，但人们对控制原位甲烷化菌生态和活动的因素知之甚少。土壤湿度、pH值、氧气、甲烷和氮水平等因素都被考虑过，尽管每一种因素都有条件地影响甲烷营养盐的多样性，但没有一种因素能对这些对环境至关重要的生物的丰度以及它们破坏甲烷的速度提供一致的解释。有趣的是，铜（Cu）是甲烷氧化菌代谢的核心，但尚未被详细研究作为影响原位甲烷氧化菌活性的因素，这令人惊讶，因为铜是颗粒甲烷单加氧酶（pMMO）的组成部分，pMMO是自然界中最有效的甲烷破坏酶。幸运的是，最近的发现提供了一个可能的解释，为什么过去对甲烷营养菌生态学的研究一直没有定论，这是基于一种叫做甲烷菌（mb）的新分子。mbs是由一些甲烷氧化菌产生的小分子，使它们能够获取、运输和使用Cu（即白团），特别是来自环境中的矿物和其他更难处理的Cu源。具体来说，数据表明mb有条件地将铜从铁氧化物和硼硅酸盐玻璃中隔离出来，这在通常不支持pMMO表达的条件下促进了pMMO的表达。因此，甲基溴的产生显然会对系统内pMMO的数量产生显著影响，并可能为产生甲基溴的生物提供竞争优势，特别是在地球化学系统中。此外，由于pMMO作为甲烷氧化剂的强度，mb介质几乎肯定会影响甲烷的净氧化活性。虽然这些观察结果很有希望，但我们对甲烷氧化菌生产mb的实际广度知之甚少，这限制了我们的预测范围；也就是说，只有一个mb被结晶（来自Methylosinus trichosporium OB3b），尽管其他三个mb已被部分表征，这表明可能存在更多的mb，但尚未被发现。因此，本项目的目的是确定产生mb样化合物的甲烷氧化菌的广度，并评估mb和其他因素如何影响地球化学背景下pMMO表达、甲烷氧化菌生态和甲烷氧化速率。初步实验将在12种不同的甲烷氧化菌上进行，以评估已知菌株和类型以及不同天然Cu条件土壤中分离物的mb产量。从这组初始mb中，将选择一个子集进行进一步纯化，以比较铜结合特性和结构与已知的M. trichosporium OB3b中的mb。在进行这些研究的同时，我们还将利用我们的模式生物——毛孢霉OB3b进行一套全面的实验，评估铜矿物学、氮源、氧水平、铁水平和其他因素对pMMO表达和甲烷氧化模式的影响。基于这些数据以及新发现的mbs的性质，将在实际土壤中进行最终实验，以校准来自确定矿物来源和从自然系统中收集的不同土壤的Cu可用性和MMO表达数据。这项工作的最终目标是提供在地球化学环境中调控MMO基因表达的最完整的图片。这将更好地为未来甲烷氧化菌的实地研究提供信息，协助气候变化研究，并为基于地球化学信息预测甲烷氧化速率提供工具。

Methane-oxidizing bacteria (methanotrophs) are nature's primary mechanism for reducing levels of atmospheric methane, the second most important greenhouse gas. Understanding the abundance and distribution of these key organisms is critical to understanding nature's response to increased anthropogenic production of the gas and other releases, such as warming-mediated melting of permafrost zones. Unfortunately, despite the importance of these organisms, little is known about what controls in situ methanotroph ecology and activity. Factors such as soil moisture, pH, and oxygen, methane, and nitrogen levels have all been considered, and although each conditionally influences methanotroph diversity, none provides a consistent explanation for the abundance of these environmentally critical organisms, nor, the rate at which they destroy methane. Interestingly, copper (Cu), which is central to methanotroph metabolism, has not been studied in detail as a factor affecting in situ methanotroph activity, which is surprising since Cu is a component of particulate methane monooxygenase (pMMO), the most efficient enzyme at methane destruction in nature. Fortunately, recent discoveries have provided a possible explanation for why past work on methanotroph ecology has been less than conclusive, which is based on a new class of molecules called methanobactins (mb). mbs are small molecules produced by some methanotrophs that allow them to acquire, transport, and use Cu (i.e., chalkophores), especially from mineral and other more refractory Cu sources in the environment. Specifically, data show that mb conditionally sequesters Cu from iron-oxides and borosilicate glass, which promotes pMMO expression under conditions that would normally not support pMMO expression. Therefore, mb production clearly makes a significant difference in the amount of pMMO within a system and likely provides a competitive advantage to organisms that produce mb, especially within geochemical systems. Further, mb-mediation almost certainly impacts net methane oxidation activity because of the strength of pMMO as a methane-oxidizing agent. Although these observations are promising, little is known about the actual breadth of mb production among methanotrophs, which limits how far we can extend our predictions; i.e., only one mb has been crystallised (from Methylosinus trichosporium OB3b), although three other mbs have been partially characterized, which suggests that more mbs likely exist, but have not yet been found. Therefore, the purpose of this project is to determine the breadth of methanotrophs that produce mb-like compounds, and assess how mb and other factors impact pMMO expression, methanotroph ecology, and methane oxidation rates in geochemical settings. Initial experiments will be performed on twelve different methanotrophs to assess mb production in known strains and types, and in isolates from soils with different native Cu conditions. From this initial set of mbs, a sub-set will be chosen for further purification to compare Cu-binding properties and structures with the one known mb from M. trichosporium OB3b. Simultaneous to these studies, a comprehensive set of experiments assessing the impact of Cu mineralogy, nitrogen source, oxygen level, iron level, and other factors on pMMO expression and methane oxidation patterns will be performed using our model organism, M. trichosporium OB3b. Based on these data and also on the nature of new mbs discovered, final experiments on real soils will be carried out to calibrate Cu availability and MMO expression data from defined mineral sources and different soils collected from natural systems. The ultimate goal of the work is provide the most complete picture yet of what regulates MMO gene expression in geochemical settings. This will better inform future field studies on methanotrophs, assist in climate change studies, and provide a tool for predicting methane oxidation rates based on geochemical information.