Nitrogen

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.

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.

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.