c-di-GMP-mediated pause behavior enables Pseudomonas aeruginosa navigation in porous environments

ABSTRACT Bacteria frequently encounter porous environments in nature and clinical settings, yet how single-flagellated pathogens navigate such confined spaces remains unclear. Here, we investigate Pseudomonas aeruginosa motility in soft agar using three-dimensional particle tracking. We discover that P. aeruginos a exhibits a distinct “pause” motility mode characterized by sustained low-speed motion with continuous reorientation in addition to its characteristic run-and-reverse swimming pattern. During pauses, flagellar filaments actively swing between adjacent pore openings while the cell body remains confined, facilitating reorientation and escape. Remarkably, bacteria pre-incubated in agar continue exhibiting pause behavior after transfer to bulk liquid, indicating that porous environments trigger lasting physiological adaptations beyond physical confinement. We demonstrate that this behavioral plasticity is mediated by elevated intracellular cyclic di-GMP levels and requires the PilZ domain effector FlgZ. These findings reveal how bacteria integrate mechanical environmental cues into regulatory circuits controlling locomotion, providing insights into pathogen behavior in clinically relevant porous environments. IMPORTANCE Bacterial pathogens must often navigate complex, confined spaces, such as tissues, mucus, and medical materials. However, how single-flagellated bacteria like Pseudomonas aeruginosa adapt their motility strategies to porous environments remains poorly understood. Here, we reveal that P. aeruginosa exhibits a distinct “pause” motility mode when encountering pore confinement, characterized by repeated reorientation events that facilitate escape. Remarkably, this behavior persists even after bacteria are returned to liquid, indicating a physiological memory triggered by the environment. We demonstrate that this adaptive response is regulated by the second messenger c-di-GMP and its effector FlgZ. Our findings uncover a direct link between mechanical environmental sensing and the molecular regulation of bacterial motility, providing new insights into how pathogens adapt to and persist in clinically and environmentally relevant porous habitats.