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

点击翻译按钮获取中文摘要

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.