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

许多矿物质，包括铁和锰的氧化物，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质在没有氧气的情况下生存，通过一个被称为“岩石呼吸”的过程将电子转移到固体金属和矿物质，这导致释放铁和锰，使它们成为生物可利用的。为了做到这一点，岩石呼吸细菌组装导电蛋白质链，穿过细胞和细胞表面的膜。越来越多的证据表明，大多数岩石呼吸细菌使用一种称为“孔蛋白-细胞色素复合物”的复合物来移动电子穿过外膜。典型的复合物由三种蛋白质组成，两种含有铁原子链的导电蛋白质称为细胞色素，还有一种类似于空桶的大孔蛋白。这种孔蛋白跨在外膜上，两种细胞色素从两侧进入桶，形成一个铁原子的导电链，允许电流从膜的一侧流向另一侧。这些孔蛋白-细胞色素复合物是允许细菌与电子设备相互作用的关键，无论是产生能量;开发活的生电生物传感器，还是直接用电能培养细菌（生电）。目前，没有可用的这些复合物的结构，限制了我们利用和适应关键结构组分（如细胞色素末端）的能力。我们也不明白如此复杂的复合物如何折叠到细胞膜中。有一个难题是，如果没有细胞色素装在里面，桶就不能组装起来，但桶只在膜中稳定，细胞色素不能进入膜。目前还不清楚桶如何在膜外组装细胞色素。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被称为希瓦氏菌的生物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，包括渗透结构的铁原子的模式（它是一条单链，还是有可以保持电荷的铁原子簇），电子如何进入/退出复合物以及什么结构特征可能有助于复合物在外膜中组装。在构建这个结构的同时，我们还将努力创建一个模型，说明复合物如何在外膜中形成。细菌膜中几乎所有的桶状蛋白都是通过一种称为BAM系统的东西组装的，该系统由许多称为伴侣蛋白的蛋白质组成，因为它们有助于蛋白质折叠。我们将产生一个希瓦氏菌突变体，其中BAM系统的组成部分在我们的控制之下，看看我们是否可以控制伴侣蛋白来控制复合物的形成。我们还将尝试从生长的细胞中分离出孔蛋白，并鉴定出可能是新的、独立于BAM的系统的一部分的任何其他分子伴侣。通过更好地理解这种跨膜导体的结构和组装，我们将能够修改复合物，使其能够被“拴系”到电极表面。组装这种可连接的复合物的基因和伴侣将被添加到模型细菌E。大肠杆菌和表达该复合物的细菌将被附着到电极上，该电极可以用于从细菌汲取电力或向细菌提供电力，最终目标是产生生物技术上重要的细菌，这些细菌可以纯粹依靠电力来饲养。

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.