Development of Bioluminescent Bacterial Bioreporters to Quantify the Bioavailability of Fe in Seawater

虽然铁在海洋系统中作为初级生产力限制剂的作用已经被广泛研究，但海洋细菌对铁可用性变化的反应仍然不太清楚。许多研究小组的研究表明，许多海洋原核生物在限制铁的生长条件下保持产生铁载体（或至少类似铁载体的化合物）的能力，因此可以在低铁可用性条件下有效地生存。虽然分析技术可以在非常小的浓度下量化总铁，并提供铁的形态和铁与有机配体的结合/解离动力学的信息，但没有不同形式的铁对自然群落的生物利用度的指标。为了了解铁在全球海洋生产力中所起的作用，需要一种基于细菌铁感应系统的工具。出于这个原因，田纳西大学诺克斯维尔分校的一名研究人员将开发、表征和校准一系列异养细菌生物发光生物报告器，这些生物报告器可以定量地响应环境铁生物利用度的变化。在构建了一系列新的报告细胞系统，并用组成型荧光蛋白标记对这些生物报告细胞进行标记（使生物报告细胞可以在天然海洋微生物群体中被枚举）之后，将在新西兰东南部亚热带辐合高营养-低叶绿素带进行田间试验，以验证本报告的田间使用，并表征天然溶解有机物铁对不同细菌的生物利用度的影响。与蓝藻中正在开发的类似工具的并排比较将提供对模型异养和光养铁获取策略之间差异的理解。目标是开发和验证一套工具集，该工具集将直接代表海洋细菌的一个子集，并作为实验室研究与所有可培养海洋细菌的比较，以及在不同地点发现的变异种群的实地研究。就更广泛的影响而言，本研究将为海洋学界提供一种生物传感器工具，能够确定铁对异养细菌群落的生物可利用性，这是了解海洋中铁的生物地球化学循环的关键组成部分。预计一旦完成，生物报告菌株将通过环境生物技术培养收集中心提供给合格的研究人员。作为该项目的一部分，两名研究生和两名本科生将在“刺激竞争性研究实验计划”（EPSCoR）机构接受培训。

ABSTRACTOCE-0526159While the role of iron as a limiting agent of primary productivity in marine systems has been extensively studied, the response(s) of marine bacteria to changes in iron availability remain less clear. Studies from numerous groups suggest that many marine prokaryotes maintain the ability to produce siderophores (or at least siderophores-like compounds) in response to iron-limiting growth conditions and as such can effectively persist under conditions of low iron availability. While analytical techniques can quantify total iron at incredibly small concentrations and provide information on iron speciation and the kinetics of iron association/dissociation with organic ligands, there are no indicators of the bioavailability of iron in different forms to natural communities. To understand the role that iron plays in global marine productivity, a tool based on the iron-sensing systems of bacteria is needed. For this reason, a researcher from the University of Tennessee at Knoxville will develop, characterize, and calibrate a series of heterotrophic bacterial bioluminescent bioreporters that quantitatively respond to changes in ambient iron bioavailability. After the construction of a series of new reporter systems, and the tagging of these bioreporters with a constitutive fluorescent protein marker (so that bioreporter cells can be enumerated in the presence of the natural marine microbial population), field tests will be carried out in the subtropical convergence high nutrient-low chlorophyll zone southeast of New Zealand to validate the field use of this reporter and characterize the effects of natural dissolved organic matter of iron bioavailability to different bacteria. Side-by-side comparisons with similar tools being developed in cyanobacteria will provide an understanding of the differences between model heterotrophic and phototrophic iron acquisition strategies. The goal is to develop and validate a tool set that will directly represent a subset of marine bacteria and serve as a comparative in laboratory studies with all cultivable marine bacteria, and in field studies with the variant populations found in diverse locations.In terms of broader impacts, this study will provide the oceanographic community with a biosensor tool capable of determining iron bioavailability to heterotrophic bacterial communities, a key component towards understanding the biogeochemical cycle of iron in the ocean. It is anticipated that once completed, the bioreporter strains will be available to qualified researchers through the center for Environmental Biotechnology culture collection. Two graduate and two undergraduate student will be trained at this Experimental Program to Stimulate Competitive Research (EPSCoR) institution as part of this project.