RoeA controls Pseudomonas aeruginosa motility via a diffusible c-di-GMP signal interpreted by the hierarchical PilZ, FlgZ, and TssZ receptors

Abstract

The second messenger cyclic di-GMP (c-di-GMP) is a central regulator of the lifestyle switch between motility and sessility in the opportunistic pathogen Pseudomonas aeruginosa. While numerous c-di-GMP metabolizing enzymes exist, the specific pathways linking individual enzymes to physiological outputs often remain unclear. Here, we elucidate a signaling pathway through which the diguanylate cyclase RoeA inhibits swimming motility. Using a combination of genetic, biochemical and phenotypic approaches, we demonstrate that RoeA synthesizes c-di-GMP to suppress motility by modulating flagellar function rather than biogenesis. A systematic screen of the eight PilZ-domain c-di-GMP receptors in P. aeruginosa identified PilZ, FlgZ, and TssZ as the essential downstream effectors. Genetic epistasis analysis revealed that these receptors function as a hierarchical module, with PilZ and FlgZ playing a primary, cooperative role. The function of all three receptors was dependent on a conserved arginine residue within their c-di-GMP binding motifs. Crucially, bacterial two-hybrid assays showed no direct physical interaction between RoeA and its downstream motility effectors. These findings support a “free diffusion-receptor capture” model, where RoeA generates a global c-di-GMP signal that is interpreted by spatially distributed PilZ-domain receptors to downregulate flagellar motor function, thus providing a key example of how signaling specificity is achieved within a global second messenger network.