The assembly and folding pathway of porin cytochrome complexes in the bacterial outer membrane

细菌外膜孔蛋白细胞色素复合物的组装和折叠途径

• 批准号: BB/P01819X/1
• 负责人: Thomas Clarke
• 金额: $ 49.36万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP01819X%2F1
• 关键词: assembly; folding; pathway; porin; cytochrome

Many minerals, including iron and manganese oxides, are broken down in the environment through bacterial action. Bacteria in the environment use this minerals to survive in the absence of oxygen by transferring electrons to solid metals and minerals through a process known as 'rock breathing', this has the result of releasing iron and manganese, making them bioavailable. In order to do this rock breathing bacteria assemble conductive protein chains that pass through the cell and across the membranes on the cell surface. There is increasing evidence that a complex known as 'porin-cytochrome complex' is used by the majority of rock breathing bacteria to move electrons across the outer membrane. The typical complex is made from three proteins, two conductive proteins that contain chains of iron atoms known as cytochromes, and a large porin protein that resembles an empty barrel. This porin straddles the outer membrane and the two cytochromes enter the barrel from each side, forming a conductive chain of iron atoms that allows electricity to flow from one side of the membrane to the other. These porin-cytochrome complexes are the key to allowing bacteria to interact with electronic devices, either to generate energy; develop living electrogenic biosensors, or directly grow the bacteria with electrical energy (electrogenesis).Currently, there are no structures available of these complexes, limiting our ability to utilise and adapt key structural components such as the cytochrome terminals. We also do not understand how such a complicated complex could fold into the membrane of the cell. There is a conundrum in that the barrel cannot assemble without the cytochrome that fits inside, but the barrel is only stable in the membrane, and the cytochrome cannot enter the membrane. It is unclear how the barrel can assemble around the cytochrome outside of the membrane. To address these questions we have performed several screening experiments and excitingly, we now have the opportunity to construct the first structural model from an organism known as Shewanella. Completing this structural model will reveal many important features, including the pattern of iron atoms that permeates the structure (is it a single chain, or are there clusters of iron atoms which can hold charge), how electrons are likely to enter/exit the complex and what structural features might assist in the complex assemble in the outer membrane. Alongside building this structure we will work to create a model for how the complex might form in the outer membrane. Almost all barrel-like proteins in the bacterial membrane are assembled through something known as the BAM system, which is composed of a number of proteins known as chaperones, as they help proteins to fold. We will generate a Shewanella mutant where components of the BAM system are under our control and see if we can controlling the chaperones will control formation of the complex. We will also try and isolate the porin from growing cells and identify any other chaperones that might be part of a new, BAM independent, system. Through a better understanding of both the structure and assembly of this transmembrane conductors we will be able to modify the complex so that it is capable of being 'tethered' to electrode surfaces. The genes and chaperones to assemble this tetherable version of the complex will be added to the model bacteria E. coli and the bacteria, expressing the complex will be attached to electrodes that can be used to either draw power from, or supply power to, the bacteria, with the ultimate goal of generating biotechnologically important bacteria that can be fed purely on electricity.

许多矿物质，包括铁和锰氧化物，在环境中通过细菌的作用被分解。环境中的细菌利用这种矿物在没有氧气的情况下生存，方法是通过一种被称为“岩石呼吸”的过程将电子转移到固体金属和矿物上，这一过程的结果是释放铁和锰，使它们成为生物可利用的。为了做到这一点，呼吸岩石的细菌组装了传导蛋白质链，这些蛋白质链穿过细胞并穿过细胞表面的膜。越来越多的证据表明，大多数岩石呼吸细菌都使用一种被称为“孔蛋白-细胞色素复合体”的复合体来移动电子穿过外膜。典型的复合体由三种蛋白质组成，两种导电蛋白质含有被称为细胞色素的铁原子链，以及一种类似于空桶的大型孔蛋白。这种孔蛋白横跨在外膜上，两种细胞色素从两侧进入桶内，形成铁原子的导电链，使电流从膜的一侧流向另一侧。这些孔蛋白-细胞色素复合体是允许细菌与电子设备相互作用的关键，要么产生能量；开发活的生电生物传感器，要么直接用电能(电生成)培养细菌。目前，这些复合体还没有可用的结构，限制了我们利用和调整关键结构组件的能力，如细胞色素终端。我们也不明白如此复杂的复合体是如何折叠到细胞膜上的。有一个难题是，如果没有适合内部的细胞色素，桶就无法组装，但桶只稳定在膜中，细胞色素不能进入膜。目前尚不清楚该桶如何在膜外的细胞色素周围组装。为了解决这些问题，我们进行了几次筛选实验，令人兴奋的是，我们现在有机会从一种名为希瓦尼拉的有机体中构建第一个结构模型。完成这个结构模型将揭示许多重要的特征，包括渗透到结构中的铁原子的图案(是单链，还是有可以保持电荷的铁原子簇)，电子可能如何进入/离开络合物，以及什么结构特征可能有助于络合物在外膜中组装。在建造这个结构的同时，我们将致力于创建一个模型，说明复合体如何在外膜中形成。细菌膜上几乎所有的桶状蛋白质都是通过BAM系统组装的，该系统由许多被称为伴侣的蛋白质组成，因为它们帮助蛋白质折叠。我们将产生一个希瓦尼拉突变体，其中BAM系统的组件在我们的控制之下，并看看我们是否可以控制伴侣将控制复合体的形成。我们还将尝试从正在生长的细胞中分离出孔蛋白，并确定任何其他可能是新的、不依赖于BAM的系统的伴侣。通过更好地了解这种跨膜导体的结构和组装，我们将能够对该复合体进行修饰，使其能够被‘拴’在电极表面。组装这种可系链形式的复合体的基因和伴侣将被添加到模式细菌大肠杆菌和细菌中，表达复合体的电极将被连接到电极上，这些电极可以用来从细菌中获取电力，或者为细菌提供电力，最终目标是产生具有生物技术重要性的细菌，这种细菌可以纯粹依靠电力喂养。

项目成果

A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

• DOI: 10.1128/mbio.02589-22
• 发表时间: 2023-02-28
• 期刊: mBio
• 影响因子: 6.4
• 作者: Norman MP;Edwards MJ;White GF;Burton JAJ;Butt JN;Richardson DJ;Louro RO;Paquete CM;Clarke TA
• 通讯作者: Clarke TA

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.

• DOI: 10.1074/jbc.ra118.001850
• 发表时间: 2018-05-25
• 期刊: The Journal of biological chemistry
• 作者: Edwards MJ;White GF;Lockwood CW;Lawes MC;Martel A;Harris G;Scott DJ;Richardson DJ;Butt JN;Clarke TA
• 通讯作者: Clarke TA