Developing a functional marker gene for Fe oxidation

铁（Fe）是地壳中含量第四丰富的元素（占质量的5%）-它的丰度和化学性质使其成为生物和环境过程中的关键元素。作为一种营养物质，铁的有效性是生态成功的关键决定因素。在土壤和含水层中，铁氧化物（铁锈矿物）隔离了许多其他营养物质和有毒金属，因此控制了生物可利用性和运输。因此，铁氧化物的形成影响了水、食物和生态资源。铁氧化物可以由铁氧化细菌（FeOB）形成，它与土壤形成、腐蚀、水净化和生物修复有关。然而，由于铁也可以非生物氧化，目前尚不清楚微生物在这些和其他环境过程中实际影响铁氧化的程度。因此，本提案的主要目标是通过确定涉及的关键基因，开发一种基于基因的微生物铁氧化测定方法。基于基因的分析，结合系统水平的理解，将允许确定FeOB活性的控制，并可能使用这些生物进行土壤和水的修复。这个项目将训练一系列学生解决地球微生物学的跨学科问题。拓展活动将包括为低收入少数族裔的5 -8年级女孩举办的年度夏令营。研究人员还将开发关于元转录组学最佳实践的新培训模块，并将实验纳入本科课程。团队将在科学咖啡馆和海岸日等场所向公众展示项目成果。最近的基因组学、蛋白质组学和超转录组学研究强烈表明，外膜细胞色素Cyc2是FeOB中广泛存在的铁氧化酶，并且还表明另一种外膜细胞色素MtoA（铁还原酶MtrA的同源物）可能起作用。当基因组或转录组中发现cyc2或mtoA时，这是否表明微生物的铁氧化能力/活性？为了回答这个问题，需要证据表明cyc2/ cyc2或mtoA/ mtoA是铁氧化的特异性。一个挑战是，FeOB分离物通常只在Fe（II）上生长，因此不可能知道哪些基因是专门表达Fe氧化的。本研究利用了一组也生长在其他电子供体上的FeOB分离物：嗜石铁氧杆菌和硫单胞菌。这些菌株中的大多数都没有很好地表征，因此研究人员将开发这些菌株作为模型，兼性FeOB来测试哪些基因在铁氧化过程中特异性表达，使用转录组学和RT-qPCR。除了cyc2和mtoA，他们的目标是确定铁氧化途径中的其他关键基因，这样他们就可以提出一套标记，他们将在富含铁的泥炭地和酸性矿井排水沉积物的环境亚转录组中进行测试。建议的工作代表了在环境中真正确定FeOB角色所需的后续步骤。一套强大的铁氧化遗传标记将使生物地球化学家更好地了解FeOB对铁循环的影响，并进一步了解它们如何将铁氧化与对水和土壤质量重要的C， N， P， S， As和其他金属的循环结合起来。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Iron (Fe) is the 4th most abundant element in Earth's crust (5% by mass) - its abundance and chemical properties make it a critical element in biological and environmental processes. As a nutrient, Fe availability is a key determinant of ecological success. In soils and aquifers, Fe oxides (rust minerals) sequester many other nutrients and toxic metals, therefore controlling bioavailability and transport. Thus, the formation of Fe oxides affects water, food, and ecological resources. Fe oxides can be formed by iron-oxidizing bacteria (FeOB), which have been implicated in soil formation, corrosion, water purification, and bioremediation. However, since Fe can also oxidize abiotically, it is currently unclear to what extent microbes actually influence Fe oxidation in these and other environmental processes. Thus, the primary goal of this proposal is to develop a gene-based assay for microbial Fe oxidation, by determining the key gene(s) involved. A gene-based assay, coupled to systems-level understanding, would allow to determine controls on FeOB activity, and perhaps use these organisms for soil and water remediation. This project will train a range of students in interdisciplinary problem solving for geomicrobiology. Outreach will include an annual camp for 5th-8th grade girls, who are low-income minorities. Investigators will also develop new training modules on metatranscriptomic best practices and incorporate experiments into undergraduate classes. The team will present project findings to the general public through venues such as Science Cafe and Coast Day.Recent genomics, proteomics, and metatranscriptomics work strongly suggests that an outer membrane cytochrome, Cyc2, is a widespread Fe oxidase in FeOB, and also suggests a potential role for another outer membrane cytochrome MtoA, a homolog of the Fe reductase MtrA. When cyc2 or mtoA is found in genomes or transcriptomes, does this indicate microbial Fe oxidation capability/activity? To answer this, there is a need for evidence that cyc2/Cyc2 or mtoA/MtoA is specific to Fe oxidation. A challenge is that FeOB isolates typically grow exclusively on Fe(II), making it impossible to know which genes are specifically expressed for Fe oxidation. This study takes advantage of a group of FeOB isolates that also grow on other electron donors: Sideroxydans lithotrophicus and Thiomonas spp. Most of these strains are not well-characterized, so investigators will develop these as model, facultative FeOB to test which genes are specifically expressed during Fe oxidation, using transcriptomics and RT-qPCR. Beyond cyc2 and mtoA, they aim to determine additional key genes in the Fe oxidation pathway, so that they can propose a suite of markers, which they will test in environmental metatranscriptomes from an Fe-rich peatland and acid mine drainage sediments. The proposed work represents the next steps required to truly determine FeOB roles in the environment. A robust set of Fe oxidation genetic markers would allow biogeochemists to better understand the influence of FeOB on Fe cycling, and further, how they couple Fe oxidation to cycling of C, N, P, S, As, and other metals important to water and soil quality.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.