Molecular analysis of how iron regime impacts ferritin A levels post-transcriptionally in E. coli

Al Hrazi, A. M. (2026) Molecular analysis of how iron regime impacts ferritin A levels post-transcriptionally in E. coli. PhD thesis, University of Reading. doi: 10.48683/1926.00128452

Abstract/Summary

Iron-storage proteins are key components of iron homeostasis. In Escherichia coli, iron is mainly stored by ferritin A (FtnA) to support growth under iron restriction. Although the process of FtnA iron uptake is well understood, mechanisms of iron release remain unclear. This study aimed to investigate the possible mechanisms that control FtnA stability and degradation, the potential role of the FtsH protease in the regulation of FtnA levels and the possibility that FtsH targets FtnA for degradation through the “N-end rule”. Furthermore, the impact of ftnA expression from a surrogate promoter on FtnA regulation was considered in order to divorce transcriptional effects from those imposed post-transcriptionally. In particular, an aim was to determine whether the increased levels of FtnA caused by an L2A/K mutation are post-transcriptionally mediated. Proteins with primary-destabilising second residues, such as FtnA, are less stable and can be targeted by proteases via the N-end rule; replacement with stabilising residues can elevate protein levels. A previous study showed that substitution of the conserved Leu2 of FtnA with Lys or Ala caused a major increase in FtnA levels, and the basis of this effect was explored here. An inducible promoter was used to drive expression of wildtype and L2A/K ftnA in mutants lacking iron-storage genes (ftnA, bfr and dps). Plasmid-dependent wildtype FtnA showed an average 3.9-fold increase in response to iron supplementation in stationary phase, and FtnA-L2A/K variants resulted in ~1.5-fold increased growth under iron restriction and much higher FtnA levels compared to the wildtype version (confirming pervious data), suggesting that the N-terminal sequence strongly influences FtnA stability and that Leu2 mediates rapid turnover. Whole-cell ICP-OES revealed an average 14.6% increase in iron for L2 variants, consistent with enhanced growth observed as a result of the increased FtnA iron-storage capacity for the L2 variants. A previous study demonstrated a potential role for FtsH in control of FtnA levels from chromosomally-encoded FtnA, and this was confirmed here: a ∆ftsH mutant showed a reproducible 3.9-fold increase in FtnA accumulation during exponential growth in response to iron. However, when ∆ftsH was introduced into a strain lacking the three iron-storage proteins and complemented with inducible ftnA (WT, L2A, L2K) from pBADrha, FtnA levels were indistinguishable with or without FtsH. This indicates that FtsH regulates FtnA mainly via effects on the native ftnA promoter not direct proteolysis (as was previously considered), and that L2A/K impacts on FtnA levels are FtsH-independent. Thus, subsequent investigation was aimed at assessing how the expression context of ftnA impacted its regulation. Therefore, the role of H-NS was examined. H-NS, known to repress chromosomal ftnA (relieved by Fe²⁺-Fur), was found instead to activate plasmid-dependent ftnA, likely through control of Prha1, with effects observed at both protein and transcript levels. RhaSR-regulated rhaT and Prha1 transcripts were also H-NS induced, suggesting that the Rha regulon is H-NS activated. Moreover, qRT-PCR showed no significant changes in plasmid-dependent ftnA transcript levels for L2 variants, confirming that elevated FtnA-L2A/K arises post-transcriptionally possibly via increased FtnA stability (reduced degradation). Such turnover may play an important role in regulating E. coli iron-storage capacity and mediating release of iron from stores.

Item Type	Thesis (PhD)
URI	https://centaur.reading.ac.uk/id/eprint/128452
Identification Number/DOI	10.48683/1926.00128452
Divisions	Life Sciences > School of Biological Sciences > Department of Bio-Engineering
Date on Title Page	November 2025
Download/View statistics	View download statistics for this item