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

海洋中光合作用固定的二氧化碳约占初级生产总量的一半。 这种初级生产的一个重要组成部分是由于海洋蓝细菌的生长，如固氮生物Trichodesmium。 例如，在热带和亚热带北大西洋，束毛藻属（Trichodesmium spp.）被认为是最重要的初级生产者。 此外，在该地区，束毛藻将最大比例的新氮引入真光层（约30 mg N/m2/天），这一数值超过了跨温跃层的硝酸盐通量。 虽然有许多生态地球化学因素可能会影响束毛藻的生产力和N2固定（如营养物质，光照，温度），最近的研究强调了P和Fe的生物利用度的至关重要性。 为了增加我们对这些元素的生物利用度如何限制初级生产和N2固定的理解，这些PI开发了用于Trichodesmium中Fe和P限制的特定分子诊断。 PI已确定P-胁迫束毛藻菌落的无菌培养物诱导酶PhoA，并且Fe-胁迫菌落表达蛋白IdiA。 PI还证明，它们可以检测田间种群中的PhoA活性和IdiA表达。在这个项目中，实验室研究将用于确定这些诊断如何与束毛藻生产力和N2固定的降低相关。 这项工作将导致两个经过充分测试的分子诊断的发展和应用，以确定是否P，Fe，或两者，限制束毛藻C和N2固定。用于种特异性PhoA活性的测定（称为酶标记荧光）使用市售试剂，并且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，以快速，轻松地确定P和Fe限制束毛藻物种从任何环境。

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.