The mechanism of the ferrous iron transport system (Feo): interaction of FeoC with the C-terminal region of FeoB

Althobaiti, O. A. (2026) The mechanism of the ferrous iron transport system (Feo): interaction of FeoC with the C-terminal region of FeoB. PhD thesis, University of Reading. doi: 10.48683/1926.00130504

Abstract/Summary

Iron is an essential element, but can also be toxic aerobically through the production of oxidizing free radicals. Two major types of iron are found in biological systems, the ferrous (Fe2+) and ferric (Fe3+) forms. The feo operon, found in many bacteria, encodes a ferrous-iron uptake pathway that functions under anaerobic conditions. The feoABC operon of Escherichia coli specifies three components: FeoA, a small cytosolic protein absolutely required for FeoB activity; FeoB, the major component consisting of an N-terminal G-protein domain in the cytoplasm and a C-terminal ferrous-permease domain embedded in the cytoplasmic membrane; and FeoC, a small Fe-S protein reported to control FeoB stability in response to oxygen and mainly only associated with the FeoAB system of facultative Enterobacteriaceae. This work consists of three results chapters. The first results chapter explores the role of FeoC on the iron-uptake activity of the Feo system by performing growth comparison of an iron uptake mutant, aerobically and anaerobically, under a range of iron-restriction conditions in the presence and absence of induced feoAB or feoABC. The results showed a clear role of FeoC in supporting iron-uptake activity under aerobic reducing conditions in the presence of DTPA for strains producing FeoAB as the sole dedicated iron uptake pathway. However, the results with single-gene DfeoA, DfeoB and DfeoC mutants did not show any distinct role of feoC in supporting Feo-dependent under iron-restricted growth aerobically; all the mutants gave similar strong growth restrictions under low-iron conditions both aerobically and anaerobically with reductant, buffer at pH 6 and chelator. Under anaerobic conditions in the presence or absence of iron, the feoAB/ABC constructs and the mutants (∆feoA, ∆feoB and ∆feoC) showed no role for FeoC in supporting FeoAB-mediated iron uptake. The second results chapter focused on the role of the C-terminal region of FeoB (which is largely unique to the Enterobacteriaceae) in supporting FeoC-dependent enhanced Feo iron-uptake activity through protein-protein interaction. The research was divided into two parts. Firstly, introduction of a series of truncations in the C-terminal region of FeoB (CFeoB) using pBADara-feoAB/ABC constructs subjected to site-directed mutagenesis. The mutants were tested under iron-restricted growth aerobically. The results illustrated that the unmutated feoABC construct provides a clear growth advantage with respect to feoAB and the vector control under aerobic iron-restricted conditions with DTPA and MES. The CFeoB truncations had an overall negative impact on Feo-dependent growth for both the FeoAB and FeoABC strains. In the second part, the CFeoB region was expressed from pBADrha in three formats; Flag tag-CFeoB; CFeoB-Flag tag; CFeoB without any Flag tag. All the Flag tag versions were transferred into the pBADara feoAB/feoABC to determine any effect of CFeoB expression on FeoAB-dependent aerobic and iron-restricted growth in the presence and absence of FeoC. The results showed that in the presence of DTPA, clear growth advantage for the FeoABC strain which indicates to the role of FeoC-enhancement of the Feo activity. The CFeoB constructs caused a reduction in the growth of the FeoABC strain which was significant in two cases. CFeoB also caused a slight overall reduction in the FeoAB-supported growth. The findings suggest that the FeoC enhancement of FeoAB dependent aerobic, iron-restricted growth can be inhibited by provision of CFeoB in Flag tagged form. This is consistent with the proposed interaction of FeoC with the CFeoB region of FeoB. In the third results chapter, the impact of FeoC (and FeoAB) on global gene expression was explored by RNAseq in the absence of iron but with ascorbate and with iron but without ascorbate, at two time points (OD 0.5. and 0.8/1.0). With ascorbate at OD 0.5, FeoAB-induced 13 genes involved in anaerobic nitrite/nitrate respiration and repressed eight and seven genes for tRNA and amino acid biosynthesis, respectively. At OD 0.8 with ascorbate, FeoAB induced nine and eight genes involved in amino acid generation and carbon catabolism, respectively, and 16 and eight genes involved in amino acid biosynthesis and nucleotide metabolism, respectively. With iron at OD 0.5, FeoAB induced 17 genes related to anaerobic energy generation and repressed 32 genes involved in combating iron restriction. At OD 1.0 with iron, FeoAB induced 12 genes involved in anaerobic energy generation and repressed 25 genes involved in combating iron restriction. The results obtained for FeoABC dependent global expression were generally similar to those for FeoAB, and together clearly indicated that feoAB/feoABC induction promotes large scale changes in the expression of genes involved in iron homeostasis in the presence of iron suggesting that Feo induction promotes excess iron accumulation resulting in enhanced Fe-Fur regulatory activity. Differential expression between the feoAB and feoABC strains allowed the effect of feoC on expression to be considered. With ascorbate at OD 0.5, FeoC induced three genes involved in carbon metabolism and repressed 13 genes involved in anaerobic energy generation, whereas at OD 0.8 three genes involved in amino acid metabolism were induced and cold shock genes were repressed. At OD 0.5 with iron, FeoC induced six genes involved in amino acid metabolism and carbon metabolism, and repressed seven and four genes involved in cysteine biosynthesis (and sulphur assimilation) and motility, respectively. At OD 1.0 FeoC induced seven genes involved in amino acid metabolism and repressed six genes associated with combating iron restriction. In summary, the results suggest that the Feo system may influence the regulation of genes involved in anerobic energy generation, in particular those associated nitrate/nitrite respiration. The impact of FeoC on Fur-dependent gene control may be related to its role in enhancing FeoAB iron uptake activity.

Item Type	Thesis (PhD)
URI	https://centaur.reading.ac.uk/id/eprint/130504
Identification Number/DOI	10.48683/1926.00130504
Divisions	Life Sciences > School of Biological Sciences > Department of Bio-Engineering
Date on Title Page	October 2025
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