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，对于了解自然界对增加的人为气体生产和其他排放的反应至关重要，例如，气候变暖导致的永久冻土带融化。不幸的是，尽管这些生物很重要，但人们对是什么控制了原位甲烷菌的生态和活性知之甚少。土壤湿度、pH值以及氧气、甲烷和氮素水平等因素都被考虑在内，尽管每种因素都会影响甲烷化微生物的多样性，但没有人能对这些对环境至关重要的生物的丰富程度或它们破坏甲烷的速度提供一致的解释。有趣的是，作为甲烷代谢中心的铜(Cu)作为影响原位甲烷代谢活性的因素还没有被详细研究，这是令人惊讶的，因为铜是颗粒甲烷单加氧酶(PMMO)的组成部分，pMMO是自然界中破坏甲烷最有效的酶。幸运的是，最近的发现提供了一种可能的解释，解释了为什么过去对甲烷菌生态学的研究一直没有定论，这是基于一种名为甲烷结合蛋白(Mb)的新分子。MBS是一些甲烷氧化菌产生的小分子，使它们能够获取、运输和利用铜(即白炭基团)，特别是从环境中的矿物和其他更难处理的铜源中获得、运输和利用铜。具体地说，数据显示，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，mb的生产显然对一个系统内pMMO的数量产生了显著的影响，并可能为产生mb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中介几乎肯定会影响甲烷净氧化活性。尽管这些观察结果很有希望，但人们对甲烷氧化菌产生mb的实际广度知之甚少，这限制了我们的预测范围；即只有一种mb被结晶(来自甲氧西林毛孢子菌OB3b)，尽管另外三种mb已经被部分表征，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类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pMMO的表达、甲烷氧化生态和地化环境中的甲烷氧化速率。最初的实验将在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来自不同自然铜条件的土壤分离株的mb产量。从这个初始的MB集合中，将选择一个子集进行进一步纯化，以将铜结合特性和结构与来自天花粉分枝杆菌OB3b的已知MB进行比较。在这些研究的同时，将使用我们的模式生物M.trichosporium OB3b进行一套全面的实验，评估铜矿物学、氮源、氧气水平、铁水平和其他因素对pMMO表达和甲烷氧化模式的影响。基于这些数据和新发现的MBS的性质，将在真实土壤上进行最终实验，以校准从确定的矿物来源和自然系统收集的不同土壤中获得的铜有效性和MMO表达数据。这项工作的最终目标是提供迄今最完整的关于在地球化学环境中调节MMO基因表达的图景。这将更好地为未来关于甲烷氧化菌的实地研究提供信息，协助气候变化研究，并提供一种根据地球化学信息预测甲烷氧化速率的工具。

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.