Development & Application of Molecular Diagnostics for Fe & P Limitation of Trichodesmium Nitrogen Fixation & Productivity

海洋中二氧化碳的光合作用固定约占初级生产总量的一半。初级生产力的一个重要组成部分是海洋蓝藻的生长，如重氮菌Trichodesum。例如，在热带和亚热带北大西洋，毛霉属(Trichodesum spp.)被认为是最重要的初级生产者。此外，在这一地区，毛霉菌向真光层引入了最大比例的新氮(~30 mg N/m2/天)，这个值超过了温跃层中硝酸盐的估计通量。虽然有许多生物地球化学因素可能影响木霉的产量和固氮(如营养、光照、温度)，但最近的研究强调了磷和铁的生物有效性的重要性。为了增加我们对这些元素的生物有效性如何限制初级生产和氮气固定的理解，这些PI开发了对毛霉中铁和磷限制的特定分子诊断方法。PI已经确定，磷胁迫下的木霉菌落的无菌培养诱导了PhoA酶，而铁胁迫下的菌落则表达了IDIA蛋白。PI还表明，它们可以检测到田间种群中PhoA的活性和IDIA的表达。在这个项目中，实验室研究将被用来确定这些诊断与毛霉菌产量和氮气固定减少的相关性。这项工作将导致两种经过良好测试的分子诊断方法的开发和应用，以确定是否P、Fe或两者都限制了毛霉菌C和N的固定。对物种特异性PhoA活性的分析称为酶标记荧光，利用商业上可获得的试剂，与在大肠杆菌中异源表达的IDIA一样，PI将向其他人提供IDIA蛋白或抗血清。这项研究的影响将是研究人员可以使用的工具，快速轻松地确定任何环境中毛霉菌的磷和铁限制。

Photosynthetic fixation of CO2 in the oceans accounts for approximately half of total primary production. A significant component of this primary production is due to the growth of oceanic cyanobacteria, such as the diazotroph Trichodesmium. For example, in the tropical and subtropical North Atlantic, Trichodesmium spp. are thought to be the most significant primary producers. Additionally, in this region Trichodesmium introduces the largest fraction of new nitrogen to the euphotic zone (~30mg N/m2/day), a value that exceeds the estimated flux of nitrate across the thermocline. Although there are many biogeochemical factors that may impact Trichodesmium productivity and N2 fixation (e.g. nutrients, light, temperature), recent studies have emphasized the critical importance of P and Fe bioavailability. To increase our understanding of how the bioavailability of these elements may constrain primary production and N2 fixation, these PIs developed specific molecular diagnostics for Fe and P limitation in Trichodesmium. The PIs have determined that axenic cultures of P-stressed Trichodesmium colonies induce the enzyme PhoA and Fe-stressed colonies express the protein IdiA. The PIs have also demonstrated that they can detect PhoA activity and IdiA expression in field populations. In this project, laboratory studies will be used to determine how these diagnostics correlate with decreases in both Trichodesmium productivity and N2 fixation. This work will result in the development and application of two well-tested molecular diagnostics for determining if P, Fe, or both, limit Trichodesmium C and N2 fixation. The assay for species-specific PhoA activity, called Enzyme Labeled Fluorescence, utilizes commercially available reagents, and as with IdiA heterologously-expressed in Escherichia coli, the PIs will provide the IdiA protein or antiserum to others. The impact of this research will be tools that can be used by investigators to quickly and easily identify P and Fe limitation in Trichodesmium species from any environment.