Regulation of dual nitrogenases in response to nitrogen stress in a cyanobacterium

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Cyanobacteria are ancient microorganisms that were among the first organisms to produce oxygen on earth. Today, cyanobacteria are important organisms in the environment. They get their energy from sunlight, their carbon from carbon dioxide in the air, and their nitrogen from the nitrogen gas in the air. They are important not only as free-living organisms, but also as partners in many symbiotic associations with plants and fungi. The cyanobacterium Anabaena can use the energy from sunlight to convert nitrogen gas in the air to ammonia. This process is called nitrogen fixation. In contrast, chemical production of fertilizer requires large amounts of fossil fuels and produces greenhouse gases. The project will investigate the regulation of nitrogen fixation in Anabaena, one of the best characterized cyanobacteria in terms of growth, physiology and genetics. A second key objective of this project is to involve, educate, and support diverse students as members of the research team. This project will include high school students, especially from underserved school districts, undergraduates and graduate students, working together as active participants in the research.Anabaena is unique in model cyanobacterial systems in that it has two homologous nitrogenase systems. One functions only in specialized anaerobic cells called heterocysts under aerobic growth condition, while the other functions only in anaerobically-grown vegetative cells. This research will determine the mechanisms that control expression of these complementary nitrogenases, focusing on global mechanisms that respond to nitrogen stress. This project will characterize the two primary activator proteins, CnfR1 and CnfR2, which lead to differential expression of the two nitrogenase gene clusters in heterocysts or vegetative cells. Further, the research will determine, at a system-wide level, the role of known global stress factors and transcription factors, in regulation of the two nitrogenase systems. Taking a synthetic biology approach, nitrogenase genes from Anabaena will be expressed in a strain that lacks this capability, as part of the longer term goal of producing a "nitroplast", a hypothetical cyanobacterial-like organelle in a plant cell that could fix nitrogen for the plant. The goal of moving nitrogenase genes to plants is to reduce reliance on nitrogen-containing fertilizers for crop production. These objectives will be achieved by combining traditional genetic techniques with synthetic biology approaches.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.