EXPLORING DNA INTERACTION AND CROSS-REACTIVITY OF RSS-SENSITIVE ArsR REGULATORS IN Vibrio cholerae AND OTHER PATHOGENS

Bacterial adaptation to H₂S-rich environments depends on transcriptional regulators capable of sensing reactive sulfur species (RSS), such as persulfides and polysulfides. Members of the ArsR family act as redox-sensitive regulators that modulate gene expression related to detoxification, stress resistance, and virulence. Vibrio cholerae has two such regulators: HlyU, which regulates genes involved in toxin expression in response to increased RSS levels, and an uncharacterized BigR-like protein, possibly involved in H₂S/RSS homeostasis, based on evidence from orthologs in other organisms.

In an effort to understand the extent to which these regulators act redundantly or selectively, we studied the DNA binding properties of HlyU and BigR from V. cholerae, and compare them to other orthologues: HlyU from Vibrio vulnificus and BigR from Acinetobacter baumannii. Ultimately, we aim to assess DNA recognition determinants within the RSS-responsive regulators that allow for functional diversity in this subfamily shown to modulate expression of toxins, biofilm formation, and RSS trafficking.

These proteins were recombinantly expressed, purified, and their thermal stability was confirmed by circular dichroism. DNA-binding affinity was determined using fluorescence anisotropy with different operator sequences. Our results show that while V. vulnificus HlyU has high affinity for its cognate operator and selectivity over orthologs operators, V. cholerae HlyU is a weaker and more promiscuous binder. AlphaFold3 and DeepPBS derived models of protein–DNA interactions provide different structural details that contribute to explain operator selectivity among these proteins by predicting key DNA-contacting residues.

Ongoing comparative analyses aim to complete the affinity measurements for cognate operators of all proteins obtained, and to determine whether exposure to RSS modulates binding affinity, ultimately helping to identify structural features associated with specific DNA recognition.