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

需要增加生物燃料和农作物的生产，以满足社会对燃料、食品和原料日益增长的需求。细胞对大气氮素的同化(固氮)需要固氮酶，这是一种只在原核生物(细菌和古生物)中存在的铁硫酶。该项目的总体目标是了解一种古生菌-产甲烷菌的固氮作用，并破译其与硫磺同化的联系。氮是作物生产中的一种限制性营养物质，在植物中进行工程固氮将减少目前用来克服这一限制的化学生产化肥所需的大量成本和能源。此外，基于固氮酶的技术是生产生物燃料的一种很有前途的策略。由高中、本科生、研究生和博士后组成的研究团队将利用现代分子和生化技术，具体确定控制甲烷菌中固氮酶硫特异性表达和组装的因素。为了了解硫磺对产甲烷菌固氮的影响范围，并将该项目与教学相结合，在PI授课的高级实验室课程中，学生将使用来自不同来源的沉积物来丰富和分离固氮甲烷菌。实验课程的学生将获得研究经验，并直接为该项目做出贡献。所有能够固氮(重氮营养)的细菌和古生菌都含有钼(Mo)固氮酶。一些重氮菌还含有钒(V)和铁(Fe)固氮酶。所有固氮酶的成熟都需要简单和复杂的铁-硫簇的输送和组装。因此，重氮菌需要感知和响应重氮化过程中有效硫和金属的变化。在重氮菌中，固氮酶中铁-硫簇的生物发生需要NIF系统，而半胱氨酸是直接的硫供体。重氮菌缺乏NIF系统，但含有ISC和SuF Fe-S簇生生物系统的成分。在铁-硫团簇的生物发生过程中，产甲烷菌也可以利用硫化物作为外源硫源和直接供硫体。对含有Mo-、V-和Fe-固氮酶的产甲烷甲烷链霉菌的初步结果表明，外源硫源(半胱氨酸或硫化物)导致固氮酶的表达、氢对重氮营养生长的抑制以及ISC和SuF Fe-S集群生物发生系统的使用方面的显著差异。因此，假设与细菌不同，同化的硫源是重氮菌固氮酶表达、组装和活性的关键决定因素。将结合遗传学、生理学、生物化学和转录组学方法对醋酸菌进行验证。具体目的是确定硫源对固氮酶表达和重氮化生长的影响，2)使用ISC和SUF系统组成的固氮酶组装，以及3)使用氢酶来缓解H2对固氮酶的抑制。该项目的综合结果预计将揭示将硫磺同化与产甲烷菌固氮联系起来的分子因素。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.