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Title: Molecular analysis of the ferrous iron transporter FeoABC in Escherichia coli
Author: Robertson, Kirstin Patricia
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2012
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Abstract:
Most bacteria have an inherent need for iron, as it is a component of various enzymes involved in vital metabolic processes. Therefore, bacteria have evolved several mechanisms to acquire iron from the environment. Bacterial iron transporters allow the active uptake of both ferrous and ferric iron. Escherichia coli is believed to possess two high-affinity ferrous iron transport systems: EfeUOB (which is induced under acidic conditions) and Feo (which is anaerobically induced). Feo consists of three proteins, FeoA, FeoB and FeoC, encoded by a three-gene operon,jeoABC. FeoB is an integral membrane protein, which is believed to act as a ferrous permease. It is a unique protein, possessing a G-protein domain - combination of a G-domain to a membrane protein has only so far been associated with higher organisms. Its role in FecB function is not clear, however it has been demonstrated that FeoB function is dependent on this domain. The role of FeoA and FeoC within the Feo system is uncertain. However, it has been suggested that FeoA may function in assisting the G-protein function of FeoB and that FeoC is a gene regulator. This study was undertaken to elucidate the function of FeoA, FeoB and FeoC and what kind of role they play in ferrous iron uptake. Firstly, it was shown that FeoA and FeoB are necessary for Feo function in E. coli, as l!.jeoA and 6.feoB mutants exhibited a poor anaerobic growth phenotype both under high and low iron availability. Raising the dosage of FeoB enhanced growth, indicating a predominant role for FeoB in Feo function. Regulatory studies with the aid of lacZ- fusions indicated that FeoC negatively regulates feoABC expression where a ~ 19-fold induction [high iron] and a ~ 10- fold induction [Iow iron] in feo-lacZ expression was observed compared to wild-type. However, expression of feo-lacZ remained iron- dependent in the feoC mutant. feoA promoter truncations were generated and studied. Results indicated binding sites for at least 3 eis-regulatory elements located between: (1) the -35 and +1 site, (2) downstream from the +1 site and (3) ~ 30 bp upstream from the -35 site. Studies using a GTPase assay confirmed that G-protein domain of FeoB has GTPase activity, which was enhanced by crude preparations of FeoA, indicating that FeoA may act as a GTPase-Activating Protein. This is the first experimental data indicating a specific role for FeoA in Feo function and a regulatory role for FeoC. ii
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.577985  DOI: Not available
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