Developing a functional marker gene for Fe oxidation

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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.