A c-di-GMP signaling network centered on the diguanylate cyclase GcbA achieves motor-specific regulation of flagellar motility in Pseudomonas aeruginosa.

Bacteria encode large, seemingly redundant repertoires of cyclic di-GMP (c-di-GMP) synthases and hydrolases, yet individual enzymes often govern discrete phenotypic outputs with high specificity. How a single diguanylate cyclase (DGC) achieves target-specific signaling remains incompletely understood. Here, we identify a multi-component c-di-GMP signaling network centered on the DGC GcbA that targets flagellar motor function in Pseudomonas aeruginosa. Genetic analysis demonstrates that GcbA-derived c-di-GMP post-translationally brakes the motor without suppressing flagellar gene expression, and GcbA physically interacts with the flagellar C-ring protein FliN, as demonstrated by bacterial two-hybrid analysis and co-purification in P. aeruginosa. A systematic two-hybrid screen of GcbA against all 40 predicted c-di-GMP-metabolizing enzymes encoded in P. aeruginosa PA14 identifies multiple interaction partners, including three phosphodiesterases (BifA, RocR, and PvrR) that counteract GcbA-dependent c-di-GMP signaling. Three PilZ-domain receptors (PilZ, FlgZ, and TssZ) mediate GcbA-dependent motility repression through genetically non-redundant pathways, yet none forms a detectable interaction with GcbA in the two-hybrid system. Genetic epistasis reveals a dissociation between bulk intracellular c-di-GMP concentration and the swimming phenotype, indicating that motor regulation by GcbA operates through a functionally distinct signal pool separable from the global cytoplasmic pool. These findings define a source-sink-sensor network centered on GcbA, in which motor-specific signaling is achieved through the physical association of the cyclase with the flagellar C-ring and its interacting phosphodiesterases, while downstream receptors independently decode the resulting c-di-GMP pool without direct contact with the cyclase.