Molecular Basis for Controlled Transmembrane Electron Transfer

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences

Abstract

Most organisms can generate energy through respiration, or breathing. While organisms such as humans are restricted to breathing oxygen, bacteria are much more flexible and can breathe a broad variety of molecules, including nitrates, sulfates and different metal oxides. During respiration, the bacteria break down different carbon compounds in their cores, and electrons are released. These electrons are then combined with a target molecule in a process known as reduction. Most of the molecules reduced during respiration are small and can easily move into the cell, however some bacteria are capable of a process known as mineral respiration, literally 'breathing rock', where the electrons are used to reduce insoluble metals. This poses a challenge because the bacteria have to safely move the electrons out of the cell and into the mineral; a process that requires electron transport through the insulating outer cell membrane. The process of mineral respiration is one of the oldest forms of respiration and alters the availability of many metals in the environment, from the essential elements iron and manganese to the extremely toxic uranium and arsenic. The levels of these metals in the subsoil and groundwater are effectively controlled by these bacteria, so understanding how they work is an important step in environmental remediation. These processes are also proving useful in the development of microbial fuel cells, where waste material could be broken down and the released electrons used either to produce electricity or to synthesis useful products such as caustic soda or ethanol.
We recently identified one of the ways in which these bacteria move electrons out of the bacteria: a biological wire made of three different protein components. Two proteins called cytochromes that contain ten iron atoms which are each held in a cofactor know as a haem. One cytochrome is found on the surface of the cell, while the other is found on the inside. The third protein forms a hollow tube in the membrane at the surface of the cell and the tips of the two cytochromes insert into this tube from opposite ends and meet in the middle, allowing electrons to move directly between the cytochromes and generating a molecular wire. The movement of electrons from the inside of the cell to the cell surface is what defines these bacteria and is essential to our understanding of how these bacteria can be utilised. This project aims to understand how these biological wires function in the membrane to move electrons across and into different minerals and electrodes. One fundamental aspect of this process that we will address is to identify whether the cytochromes are specifically tailored to different minerals, or if they simply discharge electrons into whatever is nearby. We will also find out what happens when the two cytochromes held across the membrane disconnect; whether electrons can still move out, or if the wire is somehow closed off. This research will use a number of techniques, including a novel method developed at the University of East Anglia specifically for studying these systems. This involves inserting the complex into an artificial membrane and measuring the movement of electrons across the two sides of the membrane. We will also find out how the different types of cytochromes interact with each other, whether they cluster together to form charged areas or spread across the cell surface, using special chemical labels that change when brought close together. Finally we will obtain structures of the component cytochromes as well as the entire complex. The outcome of this research will be an understanding of the most important properties of this molecular wire, which could lead to its utilisation in a variety of bioremediative and biotechnological roles.

Technical Summary

Many bacteria are capable of using insoluble metal oxides such as Fe(III)oxides and Mn(IV)oxides as terminal electron acceptors during respiration. This process of mineral respiration has an important environmental role in regulating the availability of different metal ions, including biologically essential iron and manganese as well as toxic uranium and arsenic. In microbial fuel cells these bacteria can also pass electrons to electrodes producing an electrical current. Central to the process of mineral respiration is the shuttling of electrons from the bacterial cytoplasm to the extracellular mineral surface. The current hypothesis is that a porin cytochrome complex functions as a biological wire passing electrons through the outer membrane. This complex consists of a surface exposed decaheme cytochrome MtrC, a periplasmic decaheme cytochrome MtrA and transmembrane porin MtrB. This MtrCAB complex is proposed to assemble so that both cytochromes enter MtrB and make contact in the centre, allowing direct electron transport to occur between the two sides of the outer membrane.
This research program aims to understand both the structural and catalytic roles of the surface exposed cytochromes, as well as the interaction between MtrAB and MtrC. Initially protein film voltammetry will be used to measure reduction of soluble metal chelates to determine whether the surface exposed cytochromes demonstrate substrate specificity or whether they reduce non-specifically. A novel proteoliposome system will be used to determine exactly how electrons are transported across the outer membrane by the cytochromes on opposite sides of the membrane, and if MtrC can be replaced by other surface exposed cytochromes. Finally a combination of biophysical and crystallographic methods will be used to obtain molecular detail on an important structure that has promise in a broad range of scientific areas ranging though biogeochemistry, microbiology and nanotechnology.

Planned Impact

This research aims to interrogate the properties of a transmembrane biological wire that provides the principle conduit for electrons to escape the bacterial cell. While this wire, composed of two cytochromes and a porin, is significant in allowing electrons to be transported out of the bacterium, it also has the potential for both specificity and control of electron transport across the outer membrane. Understanding the specificity and regulatory properties of this wire can be extrapolated to other porin-cytochrome complexes and greatly aid our understanding, and ultimately our ability, to exploit these processes.
Beneficiaries of this research include a diverse range of people, including scientists, biotechnologists and engineers from a variety of scientific disciplines. The properties of this biological wire will be of immediate importance to microbiologists and biochemists as they uncover the mechanisms by which bacteria survive in anoxic conditions. Geochemists and biogeochemists will be able to use this research to identify how bacteria affect the content of minerals such as iron and manganese oxides in the environment. Due to the importance of this system in several transition element cycles, it will also influence other inorganic cycles, such as the nitrogen and sulfur cycles and as such will be of long term importance to environmental scientists who seek to understand how bacteria can effect global elemental cycles. The process of mineral respiration is an ancient and complex respiration pathway that predates aerobic and other anaerobic pathways, and consequently is likely to have shaped the evolution of ancient bacteria and the composition of the early earth.
Engineers in the mining industry are interested in using mineral respiring bacteria for biomining and in the remediation of acid mine drainage. In the long term, global plans to colonise planets such as Mars will require the use of technology that alters the planets environment, and bacteria that utilise these porin-cytochrome complexes to alter the composition of different martian environments could play an important role in this process.
Many of the bacteria that employ this porin-cytochrome system are responsible for the corrosion of metals in sub-soil and aquatic environments. This research therefore is also of immediate importance to engineers and the oil industry as these bacteria contribute to the dissolution of subsurface or submarine oil pipes. Shewanella metabolism could have several important biotechnological impacts since there is potential for using mineral oxide respiring bacteria in bioremediation processes for the clean up of environments contaminated with toxic organic pollutants, for example oil leaks, or radioactive metals, such as Uranium (VI).
Metal reducing bacteria have also been explored for their potential in bioremediation using Fe(III) as electron acceptors. In a microbial fuel cell (MFC) bacteria extract electrons from organic substrates and pass them to electrodes such that an electrical current is produced. The Shewanella that express these porin-cytochrome complexes are capable of passing electrons to the electrode without the need for artificial electron donors. There is also offers the tempting possibility of supplying electrons to power different reactions within the bacteria, and researchers in different countries are currently looking at ways to adapt electrodes to optimise this process.
The work has international out-reach through our collaborations with researchers in the Pacific Northwest National Laboratory, Richland, USA that have evolved out of a US Department of Energy Biogeochemical Grand Challenge. Researchers working in the UEA laboratories benefit from these international interactions through regular video conferences, meetings and working visits to the laboratories of the PNNL.

Publications

10 25 50
 
Description This grant has so far solved the structures of two new proteins from the surfaces of an environmental bacteria, and also solved a scientific debate on the roles of flavins in cytochromes.
Exploitation Route We will now look to solve the structure of the flavin-cytochrome complex and determine why a flavocytochrome is necessary on the cell surface.
Sectors Energy,Environment

 
Description As part of an educational resource pack for Biology A-level teachers.
First Year Of Impact 2015
Sector Education
Impact Types Societal

 
Description BBSRC
Amount £476,441 (GBP)
Funding ID BB/P01819X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 12/2020
 
Title Proteoliposome model systems for studying microbe-mineral electron transfer 
Description A method of assembling lipsomes containing a redox active dye and transmembrane electron transfer complexes has been developed. These liposomes allow the study of electron transport through membranes and measurement of the interactions between cell surface proteins and insoluble metal species. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Evidence for interactions directly between microbes and minerals, the discovery of a the first porin-cytochrome electron transfer complex in geobacter sulfurreducens that allows electron transfer across the membrane. 
 
Title MtrC 
Description Structure of outer membrane cytochrome MtrC 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Allowed molecular modelling studies to begin resulting in electron transfer and flavin binding models becoming available. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4lm8
 
Title OmcA 
Description Crystal structure of outer membrane cytochrome OmcA 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact allowed other researchers to model interactions and electron transfer through this outer membrane cytochrome 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4LMH
 
Description Ines Pereira 
Organisation New University of Lisbon
Country Portugal 
Sector Academic/University 
PI Contribution Hosted a research scientist from IQTB for 2 months. Training for proteoliposome experiments.
Collaborator Contribution Research scientist awarded internship grant for travel, subsistence and bench fees while here.
Impact Publication in Nature communications on sulfate respiration.
Start Year 2015
 
Description Role of microenvironment and transition zones in subsurface reactive contaminant transport 
Organisation U.S. Department of Energy
Department Pacific Northwest National Laboratory
Country United States 
Sector Public 
PI Contribution This collaboration involved the development of techniques to study electron transfer across membranes through outer membrane cytochromes complexes. We developed the technique at UEA, which was then used by the PNNL with guidance from us to look at a range of different systems. The partnership resulted in an extension of funding from the PNNL to continue work until july 2016
Collaborator Contribution The work was performed at PNNL, using their equipment and scientists, but with the advice and support from the UEA team.
Impact doi: 10.1016/j.gca.2015.03.039 doi: 10.3389/fmicb.2015.00332 doi: 10.1111/1758-2229.12204
Start Year 2014
 
Description Confidential meeting with the company Croda 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Discussed Confidential processes undertaken by the company Croda, including potential discussions for optimisation of current processes.
Year(s) Of Engagement Activity 2016
 
Description FEMS microbiology conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talk was discussed susbtantially and led to further discussion afterwards.

After talk a Portugese scientist discussed the possibility of collaborating on a proteolipsome project they have European funding for.
Year(s) Of Engagement Activity 2015
URL http://fems-microbiology.kenes.com/
 
Description Geomicobiology society 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Supporters
Results and Impact Discussion has lead to the establishment of a Geomicrobiology Network.

After the talk a symposium was arranged and the geomicrobiology network was established.
Year(s) Of Engagement Activity 2013,2014
URL http://www.minersoc.org/gmb.html
 
Description Gordon research conference (Water) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Gordon research conferences (Environmental Sciences: water, Environmental Sciences in a Human-Impacted World) My invited talk sparked questions and significant discussion afterwards.

After my talk several international groups came to discuss several possibilities for future scientific experiments and collaborations.
Year(s) Of Engagement Activity 2014
URL http://www.grc.org/programs.aspx?year=2014&program=envsciwat
 
Description Media interest (Microbe magazine) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Article stimulated debate on the potential applicatoins of bioelectrical cells.

After publication, contacted by magazine subscribers interested in the potential applications. Also contacted by Dupont chemicals about possible applications in future.
Year(s) Of Engagement Activity 2013
URL http://www.microbemagazine.org/index.php?option=com_content&view=article&id=6382:membrane-proteins-o...
 
Description 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 stand introducing research into mineral reduction and microbial fuel cells was prepared for the Royal Norfoik Show.
Year(s) Of Engagement Activity 2016
 
Description Teaching pack 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact A case study was prepared for an A-level teaching pack entitled 'Bio batteries: makeing energy from Waste' ISBN number 9781447977452 as part of OCR A level Biology A Teacher Resource Pack 2.
Year(s) Of Engagement Activity 2015
 
Description UEA Open day demonstration 
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 Approximately 300 prospective undergraduates and parents attended a series of open days where we ran a demonstration of microbial electricity. Many students expressed interest in learning more about how microbes can produce electricity, dissolve minerals, and form wires.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description USAF focussed meeting (University of Southern California) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Discussion has led to a working document to facilitate possible research relevant to the US airforce

After the discussion we have now formed links with several US groups who are interested in collaboration.
Year(s) Of Engagement Activity 2014