Nitrogen

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

Nitrogen is most often the limiting nutrient to terrestrial ecosystems. Multiple bacterial
and archaeal lineages have evolved the ability to fix atmospheric nitrogen, representing significant inputs to agronomic systems. This reaction is carried out by nitrogenase and is driven by ATP. All known nitrogenases are inhibited by oxygen, leading to specialized adaptations that limit intracellular free oxygen levels. There is one exception: an oxygen insensitive nitrogenase has that was isolated from a Streptomyces thermoautotrophicus found in soil above burning charcoal fires. This superoxide-dependent nitrogenase was partially characterized but the strain is no longer available. The present project seeks to re-isolate the original strain, as well as seek other O2-tolerant nitrogen-fixing strains from similar environments. These strains will then have their genomes sequenced and the biochemical and physiological properties of their nitrogenases studied in order to understand the O2 tolerance and to facilitate their transfer to other organisms.

The discovery of the oxygen-tolerant superoxide-dependent nitrogenase in Streptomyces thermoautotrophicus was a remarkable result. It features in textbooks and reviews and yet there have been no primary publications on the subject since the original description in 1997. There is a good deal of speculation on why this is case, perhaps the most likely is that organism has been lost because of the difficulty of culturing cells in a carbon monoxide atmosphere. The rediscovery of this enzyme and/or other unknown enzymes with similar oxygen-insensitivity, makes this a high-risk but high-reward project. Solving the mystery of the lost enzyme is in itself a worthwhile
intellectual task. If the outcome is positive and the O2-tolerant enzyme is re-isolated and/or other examples are discovered, this will open up important new avenues of research. Thus, this modest detective project could not only trigger a serious reappraisal of mainstream thinking about nitrogenase but also would transform major aspects of biological nitrogen fixation research.

The rediscovery of the superoxide dependent nitrogenase and/or the discovery of
novel oxygen-tolerant nitrogenases, followed by their genetic, biochemical, structural, and physiological characterization, is the ultimate goal of this project. We shall exploit the complementary expertise and the transatlantic nature of our new collaborative team, which was assembled at the Nitrogen 'Ideas Lab', to make a serious attempt at this bold endeavor. This high risk, high reward project is thus well-suited to this type of program call. If successful, this work has the potential to transform all areas of nitrogen-fixation research with both biological and industrial spin-offs. In particular, this would represent the first step towards the future goal of moving oxygen-tolerant nitrogenase systems into crop plants to alleviate terrestrial nitrogen limitation in a sustainable manner without negative environmental consequences.

Technical Summary

A plant that can fix its own nitrogen would require less fertiliser, diminishing the use of fossil fuels and environmental harm. Standard nitrogenases are sensitive to O2: this fact dominates nitrogenase research. However, there is a singular example of an oxygen-tolerant nitrogenase-the superoxidedependent nitrogenase system. It has not been followed up however, possibly because of the loss of the host organism and the difficulty of culturing cells in an atmosphere of carbon monoxide. The goal of the current project is the re-isolation of the original species and/or other entirely unknown O2- tolerant nitrogen fixing systems-a high-risk but high-reward endeavor. If successful, this work will result in a serious reappraisal of mainstream thinking about nitrogenase and has the potential to transform field of nitrogen fixation. There is a vast array of potential applications for an O2
insensitive nitrogenase, not least of which is its introduction into crop plants. The result of the present project should lift the overarching constraint on the field imposed by the O2 sensitivity of nitrogenase, thereby enabling new applications not only to agriculture but also the introduction of diazotrophy to other plant, microbial and ex vivo settings.

Planned Impact

The Green Revolution succeeded in large part due to the increased use of industrially produced nitrogen fertilizers, which uses vast amounts of energy, up to 5% of all fossil fuel use, to convert atmospheric nitrogen gas into biologically usable ammonium. In essence, agriculture shifted from a system that converts contemporary solar energy into food to a system that converts ancient solar energy (in the form of fossil fuel) into food. This is clearly not sustainable. It also generates severe environmental problems through the runoff of chemical fertilizers into aquatic ecosystems. A plant that can fix its own nitrogen would require less fertiliser, use less fossil fuels and cause less environmental harm. Standard nitrogenases are sensitive to oxygen: this fact is widely accepted and underlies much of nitrogenase research. The energy used in removing oxygen gas to allow nitrogenase to function is often more than that needed to drive the already energy intensive breaking of the ultra-stable triple bond in dinitrogen. The superoxide dependent nitrogenase system that is the focus of this study is not oxygen sensitive. There is a vast array of potential applications for an oxygen-insensitve nitrogenase, not least of which is its introduction into crop plants. The result of the present project should lift the overarching constraint on the field imposed by the oxygen sensitivity of nitrogenase. This will open up new applications not only to agriculture but also allow nitrogen fixation to be engineered to other plant, microbial and ex vivo settings. A wide range of scientific disciplines and academic institution are involved in this project. The multidisciplinary team spans the fields of biophysics, biochemistry, molecular genetics, and evolutionary genomics. The training of the next generation of scientists is central to the team and the project. The project will benefit higher education through intensive research training at the undergraduate, graduate and postdoctoral levels and through international exchange between the US and UK collaborators, major scientific journals and presentations at appropriate scientific meetings. Project leaders will present public seminars and podcasts (e.g., http://wwwf.imperial.ac.uk/imedia/itunes_ collections/view/special-lectures; http://variationselectioninheritance.podbean.com/). A
project blog documenting collecting trips and research findings will be written with a general public audience in mind.
 
Description The grant was to investigate the existence of an oxygen-tolerant nitrogenase, first reported in 1997. We have shown to our satisfaction that the described nitrogenase doesn't exist, and can be attributed to contaminating organisms and artefact (MacKellar et al, 2016). We investigated the oxygen protection mechanisms of the existing nitrogenases, leading to a crystal structure of the FeSII oxygen protective protein (PDB:5FRT). Over the last years of the project completed new study on a Nitrogenase protective protein. We hope this will be published soon. A second study on a different nitrogenase protection enzyme led to us solving its structure and by a combination of spectroelelctrochemistry and enzymological approaches we came up with plausible mechanism for it function. These projects hit the secondary targets set after the first targets were reached.
Exploitation Route The non-existence of the oxygen-tolerant nitrogenase changes the text books and prevents others wasting their time by working on it. Our work on oxygen protective mechanisms of normal nitrogenase has potential biotech applications in nitrogen fixation.
Sectors Agriculture

Food and Drink

Energy

Environment

 
Description Our paper showing that the literature report of an O2 tolerant Nitrogenase was in correct had a significant impact. It is no longer routinely cited as existing as another unexplained type of nitrogenase. We have received emails from researchers who were wasting their effort working on the "oxygen-tolerant nitrogenase", who have stopped working on it as a result of our work.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Education
Impact Types Cultural

Societal

Policy & public services

 
Description Maren Friesen Department of Plant Biology, Michigan State University 
Organisation Michigan State University
Department Department of Plant Biology
Country United States 
Sector Academic/University 
PI Contribution With Dr James Murray Imperial college in our team, we Initiated the study of the "lost" strain of bacteria reported to perform O2 tolerant N2 fixation. Did the background study on the literature and on theses. Deduced the origin of the problem based on sequence comparisons. Coordinated the operation and brought together several research teams from across the world to resolve the problem.
Collaborator Contribution Partner in NSF/BBSRC Nitrogen ideas lab. Isolated new strain of bacteria from coal-seam fire soil and sequenced it.
Impact One publication in Scientific Reports resolving the long running mystery. James Murray in our team gave a plenary lecture on this in 2016 in USA
Start Year 2012
 
Description 2 Visit from Honors students from Radboud University Nijmegen 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact An interdiciplinary team of 6 honours students (biologists, enineers, physicists, chemists) plus their tutor approached us to discuss a mock research proposal in the area of photosynthesis: in 2015 on qunatum coherence in photosynthesis, in 2016 on bioinspired artifical photosynthesis. We incorporated the visit into a lab meeting with presentations form our team and the visiting students followed by a long and detailed debate. The mock grant request was made on which we gave feedback. The students, tutors and organisers we very happy with the visit and the experience of the debate with experts. This was in part the reason they came back again this year. The students themselves go on to present their (greatly updated) project to their full class interdicpilinary classes back in the Netherlands. We consider that we had a strong impact on the training aspect as well as the eventual projects.
Year(s) Of Engagement Activity 2015,2016
 
Description Advising government of transport policy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Provided arguments and citations relevant to Government policy on transport fuels with regard to biofuels.
Year(s) Of Engagement Activity 2014,2015
 
Description BBC science frontiers 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A BBC radio programme on nitrogen fixation, broadcast on radio 4 and the World Service, featuring interviews with Prof. Rutherford and Dr. James Murray on the possible oxygen tolerant nitrogenase.
Year(s) Of Engagement Activity 2013,2014
URL http://www.bbc.co.uk/programmes/b03k21p5
 
Description Debate at a International workshop on land usage at an EU Climate KIC event at Imperial College 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact A meeting of policy makers, governments representatives, academics, farmers unions, economists, geographers, members of the IPCC, etc on the development of a land use tool as part of the EU climate cick program. Rutherford was invited as a discussant and played an active role in the debate on the feasibility and energy balance questions concerning biofuels.
Year(s) Of Engagement Activity 2014
 
Description Febin Varghese talk at ECNF conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk by Febin Varghese at the 20th International Congress on Nitrogen Fixation "Azotobacter vinelandii cytochrome b': a novel flavocytochrome with a probable role in oxygen protection of the iron-only nitrogenase"
Year(s) Of Engagement Activity 2017
URL https://www.20icnf.congresosgestac.com/es/
 
Description Imperial Fringe: Lit Up 2015 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact An evening public engagement event, showcasing a variety of research activities at Imperial College. Dr Andrea Fantuzzi, Department of Life Sciences delivered a presentation describing how to 'Extract chlorophyll from algae and find out how this powerful molecule that turns light into plant matter can used to make clean energy.'
Year(s) Of Engagement Activity 2015
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/eventssummary/event_27-1-2015-11-0-39
 
Description Invited lecture at a meeting Aviation 2050 at the Bauhaus for Aviation in Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Rutherford gave a talk at the 10 year anniversary meeting of the Bauhaus for Aviation: on Aviation 2050. The aviation industry plans its expansion based on increasing use of biofuels, a policy that is backed by legislation. Rutherford gave a talk on the feasibility of these plans based on energy accounting. This had a major impact on the audience, mainly from the aviation industry.
Rutherford also talked a lot with younger members of the Bauhaus policy division and urged them to debate the proposition that a low carbon future that must mean less aviation. These debates have now started and there has interesting feedback from there.
Year(s) Of Engagement Activity 2015
 
Description Member of the Senses about Science Panel on Plants 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I answer questions associated with energy and photosynthesis posed by members of the general public. These are published on line. I have done two of these in recent years: one on chlorophyll as a sun screen and one on photosynthetic animals.
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Panel member on debate on future of UK electricity generation: Energy Future Lab/Grantham Institute 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact A panel of 4 experts gave their take on the future of UK electricity supply. As one of these experts Rutherford provided detail arguments concerning the feasibility and scaling photosynthetic biofuels based on energy accounting.
Year(s) Of Engagement Activity 2015
 
Description Participation on policy discussion on sustainable agriculture: A4S accounting for sustainability 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was invited to this 10 year anniversary celebration of the Prince of Wales charity "accounting for sustainability". I met several influencial people in the accounting industry and provided information concerning the viability of biofuels.
Year(s) Of Engagement Activity 2014
 
Description Participation on policy discussion on sustainable agriculture: the insurance industry 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I have been invited to take part in discussions among policy makers from, insurance companies, actuaries, representatives from government and the city on sustainable agriculture. I have duly provided information on biological energy issues.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description Poster at 20th International Congress on Nitrogen Fixation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster by James Murray "Structural basis of nitrogenase conformational protection from oxygen in Azotobacter vinelandii" presented at the 20th International Congress on Nitrogen Fixation in Granada, Spain.
Year(s) Of Engagement Activity 2017
URL https://www.20icnf.congresosgestac.com/es/
 
Description Provided advice on request on the feasibility of biofuels for transport to an organisation involved in setting up green bonds as finacial instruments 
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 Through contact in sustainable agriculture I was asked to provide information on the feasibility of photosynthetic biofuels to inform the process of setting up "green bonds" as finacial instruments.
Year(s) Of Engagement Activity 2014
 
Description Provided advice on request on the feasibility of biofuels in the aviation industry to a avion industry think tank 
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 In discussions about sustainable agriculture I met a member of a panel set up to advise the aviation industry on sustainable aviation. I was susbsequently invited to provide evidence on the feasiblity and scaling of biofuels.
Year(s) Of Engagement Activity 2014
 
Description nitrogenase congress, California 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact James Murray was invited to give a plenary talk at this meeting discussing our findings. We presented data that showed that the oxygen-tolerant nitrogenase was extremely unlikely to exist. This finding was discussed at the talk and over the conference with members of the nitrogenase community.
Year(s) Of Engagement Activity 2015
URL https://sites.google.com/a/ucdavis.edu/icnf/