The Pseudomonas aeruginosa Membrane Histidine Kinase BqsS/CarS Directly Senses Environmental Ferrous Iron (Fe ²⁺ )

Canonical 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 Pa BqsRS stimulus is Fe ²⁺ , but Pa BqsS has not been characterized at the protein level, and a direct interaction between Fe ²⁺ and Pa BqsS has not been demonstrated. In this work, we biochemically and functionally characterize intact Pa BqsS, 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 Pa BqsS binds a single Fe ²⁺ ion per protein dimer within the periplasmic sensor domain by using a unique ligation motif comprising Glu ⁴⁸ and Asn ⁴⁹ . Using activity assays, we show that both intact Pa BqsS and its truncated cytosolic domain have competent ATPase activity, consistent with predicted function. Importantly, we show that the ATP hydrolysis of intact Pa BqsS is stimulated exclusively by Fe ²⁺ , revealing metal-based activation of a functional, intact membrane HK for the first time. Taken together, this work uncovers important structural and biochemical properties of an intact, metal-sensing membrane HK that could be leveraged to target the BqsRS system for future therapeutic developments.