The Di-iron RIC Protein (YtfE) of Escherichia coli Interacts with the DNA-Binding Protein from Starved Cells (Dps) To Diminish RIC Protein-Mediated Redox Stress

Abstract

The RIC (repair of iron clusters) protein of Escherichia coli is a di-iron hemerythrin-like protein that has a proposed function in repairing stress-damaged iron-sulfur clusters. In this work, we performed a bacterial two-hybrid screening to search for RIC-protein interaction partners in E. coli. As a result, the DNA-binding protein from starved cells (Dps) was identified, and its potential interaction with RIC was tested by bacterial adenylate cyclase-based two-hybrid (BACTH) system, bimolecular fluorescence complementation, and pulldown assays. Using the activity of two Fe-S-containing enzymes as indicators of cellular Fe-S cluster damage, we observed that strains with single deletions of ric or dps have significantly lower aconitase and fumarase activities. In contrast, the ric dps double mutant strain displayed no loss of aconitase and fumarase activity with respect to that of the wild type. Additionally, while complementation of the ric dps double mutant with ric led to a severe loss of aconitase activity, this effect was no longer observed when a gene encoding a di-iron site variant of the RIC protein was employed. The dps mutant exhibited a large increase in reactive oxygen species (ROS) levels, but this increase was eliminated when ric was also inactivated. Absence of other iron storage proteins, or of peroxidase and catalases, had no impact on RIC-mediated redox stress induction. Hence, we show that RIC interacts with Dps in a manner that serves to protect E. coli from RIC protein-induced ROS. IMPORTANCE The mammalian immune system produces reactive oxygen and nitrogen species that kill bacterial pathogens by damaging key cellular components, such as lipids, DNA, and proteins. However, bacteria possess detoxifying and repair systems that mitigate these deleterious effects. The Escherichia coli RIC (repair of iron clusters) protein is a di-iron hemerythrin-like protein that repairs stress-damaged iron-sulfur clusters. E. coli Dps is an iron storage protein of the ferritin superfamily with DNA-binding capacity that protects cells from oxidative stress. This work shows that the E. coli RIC and Dps proteins interact in a fashion that counters RIC protein-induced reactive oxygen species (ROS). Altogether, we provide evidence for the formation of a new bacterial protein complex and reveal a novel contribution for Dps in bacterial redox stress protection.

Iron-sulfur cluster repair contributes to Y. pseudotuberculosis survival within deep tissues

“…Upregulation of ytfE following hydrogen peroxide-mediated damage of Staphylococcus aureus indicates that YtfE may play a broad role in repair, instead of just a response to nitrogen stress (44, 45). Additional studies in E. coli also suggest that YtfE repairs Fe-S cluster proteins damaged by hydrogen peroxide (13, 46-49).…”

Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues

“…Additional studies in E. coli also suggest that YtfE repairs Fe-S cluster proteins damaged by hydrogen peroxide (13, 46–49).…”

Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

“…The mononuclear iron YtfE-E159L mimics an YtfE protein following iron release and its structure shows that the process implies the movement of one of the axial histidines that exposes the metal site cavity to the exterior, thus making possible the re-entry of another iron atom to rebuild the di-iron center. Since we previously showed that YtfE interacts with E. coli Dps (DNA-binding Protein from Starved cells; Silva L.S.O. et al, 2018 ), we may hypothesize that this iron storage protein constitutes a good candidate to promote the reconstitution of the di-iron cluster in YtfE.…”

“…After 5 h, the cross-linking agent formaldehyde (1% final concentration) was added. Incubation proceeded at 37°C for 20 min, and the reaction was stopped by incubation with 0.5 M glycine at room temperature for 5 min ( Silva L.S.O. et al, 2018 ).…”