Distinct roles of PilI and ChpC in Pseudomonas aeruginosa Chp chemosensory array architecture.

Bacteria integrate environmental signals through chemosensory systems to coordinate chemotaxis and make lifestyle decisions, such as surface attachment and biofilm formation. The core sensory complex consists of three essential components: chemoreceptors, histidine kinases, and adaptor proteins. Together, these proteins form extended arrays that result in efficient signal transduction. The Chp chemosensory system of Pseudomonas aeruginosa regulates two outputs: direct actuation of type IV pili resulting in twitching motility and modulation of cyclic-AMP production, which regulates pilus biogenesis and acute virulence. A fully functional Chp chemosensory system requires two adaptor proteins, PilI and ChpC. In this study, we investigated the distinct contributions of these adaptors to chemosensory array organization. Using bacterial two-hybrid and fluorescence microscopy, we show that PilI interacts with the histidine kinase and is essential for polar signaling array formation. In contrast, ChpC fails to show direct interaction with the histidine kinase, and the loss of ChpC reduces the number of detectable Chp signaling arrays. Bioinformatic and AlphaFold modeling support the concept of distinct interactions within the array. We integrated these results with previously published data on the roles of the adaptors in signal transduction to propose a model for extended array architecture in which PilI acts as the primary adaptor, while incorporation of ChpC appears to enhance array organization and signaling efficiency.IMPORTANCEBacteria rely on chemosensory systems to detect environmental cues and regulate behaviors such as surface attachment and motility. While many of these systems use a single adaptor protein, others encode multiple adaptors whose roles in array organization and signaling are not well understood. In this study, we show that the two adaptor proteins of the Pseudomonas aeruginosa Chp system are distinct, with PilI required for assembly of signaling arrays, and ChpC appears to extend the array and its performance. These findings provide a framework for understanding how multi-adaptor chemosensory systems are structured and how array architecture influences signal integration. More broadly, this work highlights how variations in core signaling components can shape bacterial responses to environmental conditions.