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April 6th, 2025
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University of Maryland, Baltimore County
biochemistry
biorxiv

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

Paredes, A.Open in Google Scholar•Iheacho, C.Open in Google Scholar•Chacon, K. N.Open in Google Scholar•Smith, A. T.Open in Google Scholar

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 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 per protein dimer within the periplasmic sensor domain by using a unique ligation motif comprising Glu48 and Asn49. Using activity assays, we show that both intact PaBqsS and its truncated cytosolic domain have competent ATPase activity, 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. 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.

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