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Mechanisms of iron storage in Escherichia coli: a molecular-genetic analysis

Salman, A. M. H. (2018) Mechanisms of iron storage in Escherichia coli: a molecular-genetic analysis. PhD thesis, University of Reading

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Abstract/Summary

Organisms must balance their iron requirement to achieve iron homeostasis. This is achieved through several mechanisms, including sequestration of excess cellular iron by iron-storage proteins. Such proteins (‘ferritins’) act as an iron source under iron restriction; they also counter redox stress imposed by excess cellular iron that might contribute to Fenton chemistry. In Escherichia coli, there are three iron-storage proteins: ferritin A (FtnA), bacterioferritin (Bfr) and the ‘DNA-protection during starvation’ (Dps). Experiments were conducted to further investigate their respective functions and to determine whether these proteins are mutually interchangeable, i.e. can one of these iron-storage proteins be used in place of the other two. This aim was progressed by first generating a triple mutant of the corresponding iron-storage genes (BW25113 ΔftnA Δdps Δbfr) and by complementing the triple mutant with inducible plasmids expressing bfr, dps or ftnA. The lack of FtnA did not increase redox stress sensitivity, however, mutants lacking Dps had a reduced capacity to withstand challenge by hydrogen peroxide. Bfr appeared to combate H2O2 toxicity, but only in the absence of Dps (in minimal medium). Growth studies showed that the triple mutant displays a major growth impairment under iron deficient conditions and that Dps can contribute to iron-restricted growth, but only when no other iron-storage protein is available. Both Bfr and FtnA contribute to the total iron stores of E. coli K-12 in stationary phase following aerobic growth in iron-supplemented M9 medium, with Dps contributing relatively little except in the absence of Bfr and FtnA. Up to 62% of cellular iron can be stored in the wildtype by a combination of Bfr, FtnA and Dps. Both Dps and Bfr (but not FtnA) proteins provided redox-stress resistance when expressed from pBADrha in the triple mutant. Bfr acted to provide an iron source that could promote iron-restricted growth whereas neither Dps nor FtnA pre-induction caused any notable growth advantage under Fe-restriction. Subsequent studies showed that the lack of any growth advantage for pBADrha-induced ftnA was due to weak FtnA levels caused by rapid turnover of the protein through an apparent ‘N-end rule dependent’ degradation. Indeed, replacement of the second FtnA amino acid (LA/K) greatly elevated the levels of FtnA, allowing a notable support of growth under iron-restricted conditions. Inactivation of the protease-encoding ftsH gene resulted in increased the levels of FtnA in the wild type. Eight iron-storage proteins from diverse species failed to complement the triple iron-storage mutant; the reason for this is unclear. Recombinant Dps was purified and used to generate polyclonal antibodies to assist determination of Dps levels in E. coli. Encapsulin (linocin M18) of the hyperthermophile Pyrococcus furiosus was overexpressed in E. coli and purified. TEM analysis showed that the recombinant linocin M18 protein forms spherical particles of 30 nm diameter apparent. The isolated linocin M18 appeared to aggregate into a high-order oligomeric structure. SDS-PAGE analysis suggested that the linocin M18 protein is generated at low level when expressed from pBADrha in the triple mutant, which resulted in enhanced hydrogen peroxide resistance upon rhamnose induction, and also appeared to provide some support growth under low iron conditions. Thus, linocin M18 appeared to partly complement the iron-storage defect of the triple mutant.

Item Type:Thesis (PhD)
Thesis Supervisor:Andrews, S.
Thesis/Report Department:School of Biological Sciences
Identification Number/DOI:
Divisions:Faculty of Life Sciences > School of Biological Sciences
ID Code:80992

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