Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.770953
Title: A potential new mechanism of active transport across biological membranes; the EfeUOB ferrous iron transport system of Escherichia coli O157:H7
Author: Norton, Ian
ISNI:       0000 0004 7655 4923
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
Abstract:
The transport of solutes across cellular membranes against concentration gradients is a fundamental process driven by energy-dependent mechanisms including ATP hydrolysis, group translocation and proton motive force. The EfeUOB system in E. coli O157:H7 is an iron transporter that consists of an unusual set of components. The system is comprised of a probable ferric permease (EfeU), a haem peroxidase (EfeB) and a cupredoxin-metalloprotease (EfeO). It seems unique as it transports ferrous iron aerobically and functions at low pH. These conditions favour ferrous iron stability by inhibiting iron oxidation. All three components are required for its function and the system is functional in E. coli O157:H7 but cryptic in E. coli K-12 due to a frame-shift mutation in efeU. To better understand the transport mechanism and biochemistry involved this study focused on the isolation and analysis of all protein components. EfeU overexpression was attempted in this study using a double-tagged vector (pET28-SBP-TEV). Subsequent western blotting using antibodies against these tags, identified bands at the approximate molecular weight of EfeU (30.3 kDa); however, further confirmation of successful expression is required. Overexpression of native EfeB identified the majority of the protein in immature form within the cytoplasm, suggesting that the Tat pathway is a rate limiting factor for EfeB maturation and export. Removal of the Tat export sequence enabled expression of fully functional, mature protein within the cytoplasm. However, purification using anion-exchange chromatography and hydrophobic interaction chromatography caused the loss of the associated haem cofactor. Upon addition of a C-terminal hexa-histidine tag, purification of the protein (retaining the majority of the haem cofactor) was achieved using an alternative purification strategy. The cupredoxin domain of EfeO, in addition to the full EfeO protein, was successfully purified in this study. The cupredoxin domain structure was analysed by NMR, and metal-binding analysis by ICP-OES identified the domain binding both Cu2+ and Fe2+/3+ in a 1:1 ratio at separate distinct binding sites, supporting the theorised role of this domain in copper binding and subsequent electron shuttling as seen in other cupredoxins, such as azurin from Pseudomonas aeruginosa. Combination of EfeO and EfeB with Fe2+ and H2O2 generated a UV-visible spectral shift indicative of a haem redox change. EfeB oxidation by H2O2 led to a shift in the haem Soret band from 404 nm to 415 nm, with slow recovery to resting state overtime, suggestive of peroxidase activity, and the addition of Fe2+ had no effect on this observed shift. Rapid reversion of this shift on addition of EfeO suggests that the combination of EfeO and ferrous iron generates reducing power necessary to return EfeB to its resting state (probably achieved through EfeO-mediated Fe2+ oxidation). This study has provided insight into a potential new mode of redox driven iron transport across membranes, which advances our understanding of iron transport mechanisms.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.770953  DOI: Not available
Share: