Collaborative research: Unravelling mechanisms of Fe oxidation using synthetic biology and biochemistry

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Iron-oxidizing bacteria (FeOB) take advantage of Earth's abundant iron for growth. They form iron oxides or rust that sequester toxic metals and nutrients. By understanding how these microbes use iron in the environment and developing tools to domesticate them, it would be possible to harness this power for bioremediation of water, wastewater, and sediment. This research would develop the ability to monitor iron oxidation in natural and engineered systems, and furthermore optimize the process using in situ or engineered organisms. This project will educate a diverse population in microbiology, biochemistry, and synthetic biology, to develop scientific creative skills and help prepare them to use microbiology to address environmental and biotechnical challenges. Outreach activities include a "Making microbes work for us!" workshop for girls in middle school, who are first-generation college-bound students, and a range of public lectures, town halls, videos, and podcasts. The overarching message is that microbes are useful, integral parts of ecosystems, which can be harnessed to meet environmental and societal needs. This project focuses on two putative iron (Fe) oxidase systems, outer membrane cytochrome-porins Cyc2 and MtoAB, from neutrophilic FeOB. The objective is to validate the Fe oxidation activity and determine differences in function, providing information required for applying these Fe oxidases in synthetic systems. Thus, the project aims are to demonstrate and characterize Fe(II) oxidation by purified Cyc2 and MtoAB; express Fe oxidases Cyc2 and MtoAB in an engineered Shewanella oneidensis chassis strain to compare and optimize function in an environmentally-relevant strain; and develop genetic tools in neutrophilic autotrophic FeOB using synthetic biology. The Fe oxidase Cyc2 and MtoAB sequences will be taken from a common freshwater FeOB, Sideroxydans lithotrophicus ES-1, heterologously expressed, and characterized for redox potential and activity under a range of environmentally-relevant conditions. In addition, a novel chassis organism, Shewanella oneidensis, will be engineered to express Cyc2 and MtoAB, then assayed for Fe oxidation activity under various conditions. In parallel, the project will also develop tools and methods to domesticate FeOB. Development of these genetic systems will enable this and other research groups to query and verify otherwise recalcitrant pathways. This work will substantiate Fe oxidase function of Cyc2 and MtoAB, and provide insight into the biochemical mechanisms and the optimal conditions of function.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.