Researcher(s)
- Angeline Jose, Neuroscience, University of Delaware
- Jasmine Parks, , University of Delaware
Faculty Mentor(s)
- Clara Chan, College of Earth, Ocean and Environment, University of Delaware
- Jessica Keffer, , University of Delaware
Abstract
Purification and characterization of metal-oxidizing proteins from Leptothrix cholodnii SP-6
Angeline Jose, Jasmine Parks, Jessica Keffer, Clara Chan
Iron is one of the most abundant metals within the Earth’s crust, found in both natural and engineered environments. Iron oxidation is a major biogeochemical process that can occur both abiotically, through processes such as oxygen exposure, or through biotic means, catalyzed by microorganisms. Leptothrix cholodnii SP-6 is a bacterium that is capable of oxidizing both manganese and iron. Its genome encodes seven multicopper oxidases (MCOs), which are known to catalyze manganese oxidation. SP-6 does not have any known iron oxidases, raising the question of how it oxidizes iron. We hypothesize that MCOs may also be used to oxidize iron, as iron and manganese have similar chemical structures. Several MCOs are highly expressed when SP-6 is grown on iron, suggesting a potential role in iron oxidation. One of these MCOs, MofA, was heterologously expressed in E. coli and purified by affinity chromatography using an encoded C-terminal Strep-tag’s binding affinity to a Strep Tactin resin. The purified protein has iron oxidation activity, which was determined by incubating samples in reduced iron under microoxic conditions at pH 6.0 and monitoring production of oxidized iron. Another MCO, MnxG, may also be involved in metal oxidation. The mnxG gene was cloned into an established expression plasmid containing an N-terminal E. coli signal peptide for localization and a C-terminal Strep-tag for purification. Expression will be monitored via SDS-PAGE and Western blotting. Cell lysates will be assayed for Mn and Fe oxidation activity, and MnxG will be purified using Strep-Tactin affinity chromatography. Discovering novel iron oxidases in L. cholodnii SP-6 will help us understand abilities of iron-oxidizing microbes in the environment. By fully comprehending the role of microbes in environmental iron oxidation, we can harness their abilities to remediate contaminants and toxins in their environment. This research was funded by UD Summer Scholars.