Deciphering the novel link between sulfur assimilation and nitrogen fixation in methanogenic archaea

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Enhanced production of biofuels and crop plants is needed to meet society's ever-increasing demand for fuel, food, and feedstocks. The assimilation of atmospheric nitrogen by cells (nitrogen fixation) requires nitrogenase, an iron-sulfur enzyme found only in prokaryotes (bacteria and archaea). The overall goal of this project is to understand nitrogen fixation in a type of archaea, methanogens, and to decipher its link to sulfur assimilation. Nitrogen is a limiting nutrient in the production of crop plants, and engineering nitrogen-fixation in plants would alleviate the substantial costs and energy needed to chemically produce fertilizers currently used to overcome this limitation. Also, nitrogenase-based technology is a promising strategy to produce biofuels. The research team of high school, undergraduate, graduate, and postdoctoral students will use modern molecular and biochemical techniques to specifically determine the factors that control the sulfur-specific expression and assembly of nitrogenase in methanogens. To understand the scope of the effect of sulfur on nitrogen fixation by methanogens and to integrate the project with teaching, students in an upper-level lab course, taught by the PI, will use sediments from diverse sources to enrich and isolate nitrogen-fixing methanogens. Students in the lab course will gain research experience and contribute directly to the project.All bacteria and archaea capable of nitrogen fixation (diazotrophy) contain molybdenum (Mo) nitrogenase. Some diazotrophs also contain vanadium (V) and iron (Fe) nitrogenases. The maturation of all nitrogenases requires the delivery and assembly of simple and complex iron-sulfur clusters. Thus, diazotrophs need to sense and respond to changes in available sulfur and metals during diazotrophy. In diazotrophic bacteria, the NIF system is required for the biogenesis of the iron-sulfur clusters in nitrogenase, and cysteine serves as the direct sulfur donor. Diazotrophic methanogens lack the NIF system but contain components of the ISC and SUF Fe-S cluster biogenesis systems. Methanogens can also use sulfide as an exogenous sulfur source and as the direct sulfur donor in the biogenesis of iron-sulfur clusters. Initial results with the model methanogen Methanosarcina acetivorans, which contains Mo-, V-, and Fe-nitrogenases, indicate that the exogenous sulfur source (cysteine or sulfide) causes profound differences in nitrogenase expression, inhibition of diazotrophic growth by hydrogen, and usage of ISC and SUF Fe-S cluster biogenesis systems. Thus, the hypothesis is that unlike bacteria, the assimilated sulfur source is a key determinant in the expression, assembly, and activity of nitrogenases in diazotrophic methanogens. A combination of genetic, physiological, biochemical, and transcriptomics approaches will be used with M. acetivorans to test the hypothesis. The specific objectives are to determine the effect of sulfur source on 1) diazotrophic growth and nitrogenase expression, 2) the use of ISC and SUF system components for the assembly of M. acetivorans nitrogenases, and 3) the use of hydrogenase to alleviate inhibition of nitrogenase by H2. The combined results of the project are expected to reveal the molecular factors that link sulfur assimilation to nitrogen fixation by methanogens.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.