# Electron Paramagnetic Resonance Spectroscopy Reveals Promoter Dependent Transcription Regulation by Copper Activated CueR in Pseudomonas aeruginosa

## TL;DR

This study uses EPR spectroscopy to show how a copper-sensitive protein in Pseudomonas aeruginosa activates different genes to manage copper stress.

## Contribution

The study reveals promoter-specific transcription regulation by PACueR using EPR and DNA spin-labeling, a novel approach for this system.

## Key findings

- PACueR activates copZ2 more strongly than mexPQ-opmE under copper stress.
- EPR spectroscopy captures PACueR conformational changes during transcription initiation.
- Promoter affinity and dyad symmetry influence copper-regulated gene expression levels.

## Abstract

Metal responsive transcription factors are essential for bacterial metal homeostasis, allowing cells to regulate metal uptake, efflux, and detoxification in response to fluctuating metal ion levels. Among these, CueR, a member of the MerR family, is widely found in Gram‐negative bacteria. While E. coli CueR has been extensively studied, revealing that it adopts multiple conformational states to regulate transcription, P. aeruginosa CueR (PACueR) remains less characterized, with no resolved structure despite regulating a broader set of genes. In this study, we applied electron paramagnetic resonance (EPR) spectroscopy combined with DNA spin‐labeling to investigate the conformational states of PACueR bound to two different promoter sequences, copZ2 and mexPQ‐opmE. We examined the effects of PACueR binding and copper addition, capturing the transcription initiation stage that represents an essential step in copper homeostasis regulation of P. aeruginosa. Our results reveal promoter‐specific differences in PACueR DNA interactions, suggesting that while the core transcription initiation mechanism is conserved, variations in promoter affinity and length of dyad symmetry fine‐tune transcription levels in response to copper. These findings highlight the value of EPR spectroscopy in probing metal‐dependent transcription mechanisms and offer new insights into copper regulation in P. aeruginosa, a clinically important pathogen.

We performed electron paramagnetic resonance (EPR) experiments to reveal that P.
aerguinosa CueR copper transcription factor enhances expression of copZ2 more strongly than mexPQ‐opmE under copper stress, consistent with their detoxification roles. Findings highlight EPR as a key tool for probing metal‐regulated transcription.© 2026 WILEY‐VCH GmbH