Biology subjects

Chacon, K. N.

Publications and source records attributed to Chacon, K. N..

3 recordsLinked to original sources

bioRxiv2025

The Response Regulator BqsR/CarR Controls Ferrous Iron (Fe2+) Acquisition in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that forms biofilms and is responsible for antibiotic-resistant hospital-acquired infections in humans. The P. aeruginosa BqsRS two-component system regulates biofilm formation and dispersal by sensing extracytoplasmic Fe2+, but the mechanistic details of this process are poorly understood. In this work, we report the crystal and solution structures of the PaBqsR response regulator receiver domain, comprising a ({beta})5 response regulator assembly, and the DNA-binding domain, comprising a helix-turn-helix motif. Consistent with its cognate stimulus being Fe2+, we show that PaBqsR binds directly to the promoter region of the feo operon that encodes the bacterial Fe2+ transport system FeoABC. Corroborating these in vitro results, transcriptional studies show that PaBqsR is a global regulator controlling many important genes in PAO1, including the feo operon. Intriguingly, promoter-based assays reveal that PaBqsR is a dynamic regulator that responds to bioavailable Fe2+, likely through the ability of PaBqsR to bind Fe2+ directly via a His-rich motif, independent of the PaBqsS membrane His kinase. This mode of regulation is unprecedented among OmpR-like response regulators but represents an important level of control over Fe2+ acquisition in P. aeruginosa that could be an attractive therapeutic target to treat hospital-acquired infections.

biochemistry↗

bioRxiv2025

The Pseudomonas aeruginosa Membrane Histidine Kinase BqsS/CarS Directly Senses Environmental Ferrous Iron (Fe2+)

Prokaryotic two-component signal transduction systems (TCSs) are widely utilized by bacteria to respond to their environment and are typically composed of a transmembrane sensor His kinase (HK) and a cytosolic DNA-binding response regulator (RR) that work together to respond to environmental stimuli. An important TCS that regulates the expression of genes involved in biofilm formation and antibiotic resistance in many pathogens is the BqsRS/CarRS system, originally identified in Pseudomonas aeruginosa. Transcriptomics data suggested that the cognate PaBqsRS stimulus is Fe2+, but PaBqsS has not been characterized at the protein level, and a direct interaction between Fe2+ and PaBqsS has not been demonstrated. In this work, we biochemically and functionally characterize intact PaBqsS, an iron-sensing membrane HK, for the first time. Using bioinformatics, protein modeling, metal analyses, site-directed mutagenesis, and X-ray absorption spectroscopy (XAS), we show that PaBqsS binds a single Fe2+ ion within its periplasmic domain containing an N/O-rich ligation sphere that includes Glu48 as a key metal ligand. Using activity assays, we show that both intact and truncated PaBqsS have competent ATPase activities, consistent with predicted function. Importantly, we show that the ATP hydrolysis of intact PaBqsS is stimulated exclusively by Fe2+, revealing metal-based activation of a functional, intact membrane HK for the first time. Moreover, stimulation assays of PaBqsS variants demonstrate the importance of Glu45 and Asn49 in the sensing and signal transduction pathway. Taken together, this work uncovers important structural and biochemical properties that could be leveraged to target the BqsRS system for future therapeutic developments.

biochemistry↗

bioRxiv2021

Iron-sulfur clusters are involved in post-translational arginylation

Eukaryotic arginylation is an essential post-translational modification that both modulates protein stability and regulates protein half-life through the N-degron pathway. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite its conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we have discovered that ATE1s bind a previously unknown iron-sulfur cluster that is conserved across evolution. We have extensively characterized the nature of this iron-sulfur cluster, and we show that the presence of the iron-sulfur cluster is linked to alterations in arginylation efficacy. Finally, we demonstrate that the ATE1 iron-sulfur cluster is oxygen sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Thus, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

biochemistry↗