Abstract 1901 Expression of acidophilic iron metal transport enzymes

The High Country of Southwest Colorado is littered with hundreds of inactive and abandoned mining sites. These abandoned mines are exposed to atmospheric oxygen and fill with water and leak out acidic water with high concentrations of soluble metals. Extremely acidophilic bacteria are widespread in these systems and the metabolic potential, ecological functions, and evolutionary history of microorganisms is not well studied. These local mine sites provide an opportunity to study unique iron-oxidizing microorganisms as they are rich in soluble iron (Fe3+) and some acidophiles are shown to have multiple iron uptake systems. Previously, 16S rRNA amplicon sequencing on sediment samples from these mine sites identified acidophilic microorganisms: Gallionella spp., "Ferrovum" spp., and Acidithiobacillus spp., and Leptospirillum spp. The Acidithiobacillus genus has multiple iron uptake systems and we performed a sequence analysis that identified genes from the Feo system in Acidithiobacillus thiooxidans (Uniprot identifier A0A543Q4R5). We have developed a workflow for the heterologous expression and purification of the soluble N-terminal domain NFeoB and FeoC metal transport enzymes. Expression testing was performed in BL21 (DE3) competent cells and both ion exchange and size exclusion chromatography were used. This work will establish interactions between NFeoB and FeoC, which isn't well-studied in acidophiles. Through this work we are developing the methods necessary for future structural studies using X-ray crystallography. The goal of this project is to provide an approach to study acidophile genomes and characterize their metals uptake systems to enable application for bioremediation methods. BLaST is supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, UL1GM118991.