Nitrogen

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Nitrogen is most often the limiting nutrient to terrestrial ecosystems. Multiple bacterialand 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 worthwhileintellectual 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 ofnovel 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.