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. 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. Functional assays reveal that both intact and truncated PaBqsS have competent ATPase activities, consistent with prediction. 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. 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.