The assembly and folding pathway of porin cytochrome complexes in the bacterial outer membrane
Lead Research Organisation:
University of East Anglia
Department Name: Biological Sciences
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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.
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.
Publications
![publication icon](/resources/img/placeholder-60x60.png)
Costa NL
(2018)
Electron transfer process in microbial electrochemical technologies: The role of cell-surface exposed conductive proteins.
in Bioresource technology
![publication icon](/resources/img/placeholder-60x60.png)
Beblawy S
(2018)
Extracellular reduction of solid electron acceptors by Shewanella oneidensis
in Molecular Microbiology
![publication icon](/resources/img/placeholder-60x60.png)
Edwards MJ
(2018)
Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.
in The Journal of biological chemistry
![publication icon](/resources/img/placeholder-60x60.png)
Tokunou Y
(2018)
Whole-cell circular dichroism difference spectroscopy reveals an in vivo-specific deca-heme conformation in bacterial surface cytochromes.
in Chemical communications (Cambridge, England)
![publication icon](/resources/img/placeholder-60x60.png)
Duarte AG
(2018)
An electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation.
in Nature communications
![publication icon](/resources/img/placeholder-60x60.png)
![publication icon](/resources/img/placeholder-60x60.png)
Edwards MJ
(2020)
The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire.
in Cell
![publication icon](/resources/img/placeholder-60x60.png)
Edwards MJ
(2020)
Role of multiheme cytochromes involved in extracellular anaerobic respiration in bacteria.
in Protein science : a publication of the Protein Society
![publication icon](/resources/img/placeholder-60x60.png)
Clarke TA
(2020)
Uncovering nature's electronics.
in Nature chemical biology
![publication icon](/resources/img/placeholder-60x60.png)
Piper SEH
(2021)
Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit.
in Frontiers in microbiology
Description | Recently finished the structure of the outer membrane cytochrome complex, called the MtrC complex. We published a low resolution structure of this in 2018, and now have the molecular detail that allows use of this as a method for electron transfer across cell membranes. The Mtr complex is now complete and has now been published. The structure has revealed many exciting new details including showing the route used to pass electric current into and out of cells, it also shows how these complexes might assemble, and how they are exposed on the cell surface. This work has selected as a highlight for the Annual UKRI diamond review. This structure is also now being used as a template to model related processes in other bacteria. We also assembled several new plasmids based around the cytochrome maturation system of Shewanella. While the genetic constructs are availible and appear to function, there is little difference in their ability to make recombinant cytochromes when compared with other systems. |
Exploitation Route | We can connect bio-electrical systems to these membranes and move electrons across the membrane. This can be useful in unbalanced fermentations, as well as in connecting cells to electrodes to be used as biosensors. The model can be used to study electron transfer in biological systems. |
Sectors | Energy Manufacturing including Industrial Biotechology |
URL | http://papers.ssrn.com/sol3/papers.cfm?abstract_id=3445677 |
Description | Mechanisms of extracellular electron transfer in shewanella have begun to be used in extracellular metal reduction and potentially in recovery of precious metals from electronic waste streams. This is evidenced by increased interactions between academics and metal foundry companies who are keen to identify different ways to reduce metals. Groups in Manchester and Edinburgh are utilising this understanding to improve metal recovery. |
First Year Of Impact | 2022 |
Sector | Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Description | Tuning extracellular cytochromes for enhanced metal recovery and nanoparticle formation |
Amount | £290,637 (GBP) |
Funding ID | BB/X011453/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 02/2023 |
End | 01/2025 |
Title | Liquid-Chromatography Mass Spectrometry Describes Post-Translational Modification of Shewanella Outer Membrane Proteins |
Description | LC-MS data for MtrCAB complexes from Shewanella oneidensis MR-1 and Shewanella baltica OS-185.LC-MS data for a soluble form of MtrC from Shewanella oneidensis MR-1 carrying a C-terminal Strep II tag. |
Type Of Material | Database/Collection of data |
Year Produced | 2023 |
Provided To Others? | Yes |
URL | https://figshare.com/articles/dataset/Liquid-Chromatography_Mass_Spectrometry_Describes_Post-Transla... |
Title | Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme using pump-probe spectroscopy. |
Description | Data from spectroscopic, electrochemical, voltammetric and computational studies as presented in van Wonderen et al 'Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme'. Data presented in the Main and Supporting Information Appendix are included. |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
URL | https://figshare.com/articles/dataset/van_Wonderen_et_al_PNAS_2021_Data_Sets_xlsx/16621714 |
Title | Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme using pump-probe spectroscopy. van Wonderen et al |
Description | Spectroscopic, electrochemical and voltammetric data desribing properties of photosensitized MtrC proteins. The data are presented as figures in van Wonderen et al 'Nanosecond heme-to-heme electron transfer rates in a spectrally unique His/Met-ligated heme', the manuscript and supporting information appendix. |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
URL | https://figshare.com/articles/dataset/Nanosecond_heme-to-heme_electron_transfer_rates_in_a_multiheme... |
Title | Structure of the Mtr complex |
Description | X-ray crystallographic model of the Mtr complex |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | First model explaining how gram negative bacteria are extracellular. |
URL | https://www.rcsb.org/structure/6r2q |
Description | Advisory board member of the Danish "Centre for Electromicrobiology" |
Organisation | Aarhus University |
Country | Denmark |
Sector | Academic/University |
PI Contribution | Involved in scientific guidance and direction of the Centre for Electromicrobiology at Aarhus university, in particular helping to understand the biochemistry and physiology of Cable Bacteria. |
Collaborator Contribution | As an advisory board member I meet with the centre director (Lars Peter Nielsen) at regular intervals and help to guide the science of this Danish centre of excellence. I also help with their annual Electromicrobiology programs in enlisting speakers. |
Impact | Outputs are not currently available. |
Start Year | 2018 |
Description | Event at Norfolk show |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A Science outreach event describing electrogenic bacteria in the environment. A range of interactive demonstrations introducing schoolchildren and the general public to environmental bacteria, bacterial physiology, energy and protein structure through VR. |
Year(s) Of Engagement Activity | 2019 |
URL | https://royalnorfolkshow.rnaa.org.uk/discovery-zone/ |
Description | Latitude festival June 2018 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A life/energy event taking place at the latitiude festival (suffolk). Approximately 1000 children and parents took part in the latitude festival display on the display 'life without oxygen' |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.latitudefestival.com/family/kids-area/wildlife-weird-science-adventure |
Description | Metal bio-recovery and bio-remediation: Contributions to the circular economy |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Participated and. presented at a metals in biology NIBB looking at ways to utilise microbes in a circular economy. |
Year(s) Of Engagement Activity | 2019 |
Description | Norwich Science Festival October 2019 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | A interactive workshop based around electrogenic bacteria. All members funded by the grant participated in this event. Activities included taking minature core samples, building a biofilm, making a conductive bacteria chain. |
Year(s) Of Engagement Activity | 2019 |
URL | https://norwichsciencefestival.co.uk/about/norwich-science-festival-2019/ |
Description | University open days |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Workshop and activities associated with grant objectives, demonstrating the current research to prospective students. |
Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019 |