The essential role of copper in bacterial methane oxidation: characterising a novel copper protein involved in storage and the soluble copper proteome

甲烷氧化细菌利用甲烷（一种强效温室气体）作为碳和能量的来源。为了使这些生物能够最有效地发挥这一关键功能，它们需要大量的铜。甲烷氧化细菌已经进化出了新的机制，以确保它们能够积累代谢甲烷所需的铜。这包括被称为甲烷菌素（mbtin）的小肽的分泌，它能够隔离存在于生物体周围环境中的微量铜。甲烷氧化细菌利用甲基锡来吸收铜，其主要用途之一是氧化甲烷的酶（甲烷单加氧酶）。这些生物是如何处理铜的，铜既具有潜在的毒性，又至关重要，尚未得到详细的研究。在分析甲烷氧化细菌Methylosinus trichosporium OB3b的提取物时，我们发现了许多高丰度的可溶性铜池，其中含有结合这种金属的蛋白质。由于只预测了两种低丰度的可溶性铜蛋白（含铜的甲烷单加氧酶是膜结合的），因此在毛孢霉OB3b中存在大量可溶性铜是出乎意料的。其中一个高丰度的铜池被发现含有一种新的铜蛋白，可能与铜离子的储存有关，这里称为Csp1。其他可溶性铜池尚未被详细研究。在提议的工作中，我们将研究铜与Csp1在毛孢分枝杆菌OB3b中的结合，并使用我们通过过表达宿主大量生产的蛋白质。我们将分析Csp1的结构以及它是如何受到铜结合的影响的。我们将通过基因工程研究毛孢分枝杆菌OB3b在铜储存中的作用，并观察生长过程中的铜水平如何影响Csp1的产生。类似于Csp1的蛋白质存在于甲烷氧化细菌和其他细菌中。从毛孢杆菌OB3b和枯草芽孢杆菌中提取的Csp1同源物将被研究，枯草芽孢杆菌是一种已经研究过铜处理的生物，对基因工程方法来说更容易处理。在M. trichosporium OB3b中发现的其他可溶性铜池也将被分析，目的是寻找其他新的铜结合蛋白。拟议的研究将深入了解铜在一个相对简单的具有环境重要性的系统中的管理，因此在帮助了解自然界中甲烷的氧化和减轻这种温室气体对全球变暖的贡献方面具有应用价值。此外，作为液体燃料和化工生产的原料，大量的气态甲烷储量尚未开发，这是由于现有的转化为甲醇的工艺成本高、难度大，而甲醇是一种很容易由甲烷氧化细菌进行的反应。因此，了解这些生物如何处理甲烷氧化的重要辅助因子铜具有潜在的生物技术应用价值。

Methane-oxidising bacteria use methane, a potent greenhouse gas, as a source of carbon and energy. To enable these organisms to perform this key function most effectively they require large quantities of copper. Methane-oxidising bacteria have evolved novel mechanisms that ensure they are able to accumulate the copper they need to metabolise methane. This includes the secretion of small peptides called methanobactins (mbtins) that are able to sequester the minute amounts of copper present in the organism's surrounding environment. Methane-oxidising bacteria use mbtin to uptake copper, with one of the main uses being for the enzyme that oxidises methane (a methane monooxygenase). How these organisms handle copper, which is potentially toxic as well as being essential, has not been studied in any detail. Whilst analysing extracts from Methylosinus trichosporium OB3b, a methane-oxidising bacterium, for mbtin we have found a number of high abundance soluble copper pools containing proteins that bind this metal. The presence of large amounts of soluble copper in M. trichosporium OB3b was unanticipated as only two low-abundance soluble copper proteins are predicted (the copper-containing methane monooxygenase is membrane bound). One of the high abundance copper pools has been found to contain a novel copper protein, potentially involved in storing copper ions, herein called Csp1. The other soluble copper pools have not yet been studied in any detail.In the proposed work we will study copper binding to Csp1 both in M. trichosporium OB3b and also using protein that we have produced in large amounts via an over-expression host. We will analyse the structure of Csp1 and how this is influenced by copper binding. We will investigate the role of Csp1 in copper storage by genetically engineering M. trichosporium OB3b, as well as by looking at how copper levels during growth influence the production of Csp1. Proteins similar to Csp1 are present in methane-oxidising bacteria and also in other bacteria. The Csp1 homologues from M. trichosporium OB3b and Bacillus subtilis, an organism in which copper handling has been studied and that is more tractable for genetic engineering approaches, will be studied. The other soluble copper pools that have been identified in M. trichosporium OB3b will also be analysed with the aim of finding additional novel copper-binding proteins. The proposed studies will provide insight into copper management in a relatively simple system of environmental importance and therefore has applications in helping to understand the oxidation of methane in Nature and the mitigation of the contribution this greenhouse gas makes to global warming. Furthermore, vast gaseous methane reserves are untapped as a feedstock for liquid fuels and chemical production due to the expense and difficulties associated with the available processes that can facilitate the conversion to methanol, a reaction readily performed by methane-oxidising bacteria. Understanding how these organisms handle an essential cofactor, copper, for the oxidation of methane therefore has potential biotechnological applications.