Purification of Feo proteins and analysis of residues important for Feo protein interactions
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Iron is an essential element for virtually all forms of life. Complicating matters, it is present in the insoluble ferric form in aerobic environments, while the more soluble ferrous form is found in anaerobic or reducing environments. Vibrio cholerae, the causative agent of the disease cholera, requires iron to survive. In order to meet the need for iron, V. cholerae expresses a variety of iron acquisition systems. One of these systems, Feo, is highly conserved among bacterial species as well as archaea and transports ferrous iron. The Feo system consists of three proteins: FeoA, FeoB, and FeoC. Previous work using the bacterial adenylate cyclase two hybrid system has shown that FeoC interacts with the cytoplasmic N-terminal domain of FeoB. However, the significance of this interaction is not known. In this study, V. cholerae Feo system proteins were analyzed for residues important for the interaction between FeoB and FeoC. In addition, FeoA and FeoC were purified for antibody production. It was found that a residue in the G protein domain of FeoB was not necessary for interaction with FeoC. However, a conserved residue in FeoC did abolish the interaction with FeoB. These results indicate that there is at least one residue important in the interaction of FeoB and FeoC, although further characterization will most likely reveal more. Antibodies to FeoA and FeoC were generated to use them for further characterization of the Feo system.