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

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.

In the absence of oxygen or other soluble electron acceptors, many micro-organisms are capable of utilising insoluble metal oxides or minerals as terminal electron acceptors during respiration. For gram-negative bacteria this requires that catabolic electrons released during oxidation of organic molecules must pass through the outer membrane to the cell surface. A complex, known as a 'porin-cytochrome complex' is used to transport electrons through the outer membrane. Several porin-cytochrome complexes have been identified so far, including the Shewanella MtrCAB complex, which is composed of two cytochromes MtrC and MtrA and a porin MtrB.
We recently obtained crystals of an MtrCAB porin-cytochrome complex. Partial phasing of this data has shown the orientation of MtrC and the positions of several MtrA iron atoms. Using a strategic approach of model building, resolution and phase improvement we will generate a structural model of this first outer membrane electron transfer complex.
There is evidence to suggest the MtrB porin orientation is reversed, and that MtrB may have an unusual transport pathway. To first determine whether the MtrAB component is assembled using the BAM complex we will generate S. oneidensis mutants where either bamA or degQ is under the control of an inducible promotor and match expression of MtrB with BamA or DegQ. We will also isolate MtrB using affinity chromatography to identify other possible chaperones in the pathway.
Finally we will exploit the structural and assembly data to improve methods of incorporating MtrCAB into genetically tractable gram-negative bacteria. We will express both the cytochrome c maturation pathway from S. oneidensis and the mtrCAB genes with any accessory chaperones on plasmid based vectors and incorporate these into Esherichia coli. Enhanced and targeted expression of MtrCAB through the E coli outer membrane will allow electrical communication with redox active periplasmic proteins.

Who will benefit:
Researchers looking to understand the fundamentals of microbe-mineral interactions will benefit from a molecular level understanding of these process of extracellular electron transfer. Other academics will benefit including biochemists, molecular biologists and microbiologists, through application of the structural data and the genetic tools developed within this grant. The UK will also benefit from training of the researcher Co-I in X-ray free electron lasers, an area where the UK currently has little expertise.
The general public will also benefit from this grant; the concept of 'biological wires and semiconductors' is simple and engaging and is a useful tool to engage with the general public about environmental bacteria, energetics and elemental cycles.
Shewanella and microbial fuel cells can be used to teach students and school children about biology and electricity and educational companies will be able to use this knowledge to improve their products. Production of proteins is an important component of the A-level syllabus and this research can be used to stimulate interest in this area, with relevance to topics 2-Biological molecules, 6-Nucleic acids and protein synthesis, 12-Energy and respiration, 19-Genetic technologies.
Fermentation scientists and biotechnologists interested in improving product yields by supplementing cultures with electrical energy will benefit from this important step towards electrogenic bacteria. In principle feeding bacteria electricity will decrease the amount of chemical energy spent on ATP and NADPH production, allowing yields to be improved.

How will they benefit.
After completion structural models will be deposited in the RCSB protein data bank and will be accessible to everyone on publication. Plasmids and Shewanella strains designed during research will be available upon request, and will provide a useful tool for those who work on the characterisation of c-type cytochromes. Protein modellers and bioinformaticians will be able to probe the complex for key structural motifs to help identifiy porin cytochrome complexes in the future. Less than 1 % of known protein structures are membrane associated and even fewer have soluble domains on both sides of the membrane. Development of methods to tether microorganisms to electrodes will help engineers and electrochemists that seek to utilise extracellular electron transfer to perform useful organic and inorganic reductive reactions.
We will engage the public through a broad range of activities, including a blog tracking the progress of the structure, an online video describing the model after publication, and a microbial fuel cell manual, aimed at helping teachers use fuel cells effectively as teaching tools. We will participate in science festivals, using 3D printed models of the porin cytochrome structure and various microbially powered devices to engage the public; these will be advanced from our current devices that are used at university open days. We will also engage with companies such as Keegotech, who make microbial fuel cells for educational purposes and have a customisable range of fuel cell devices.