Genetic Dissection of Cyclic di‐ <scp>GMP</scp> Signalling in <scp> <i>Pseudomonas aeruginosa</i> </scp> via Systematic Diguanylate Cyclase Disruption

The second messenger bis-(3' → 5')-cyclic dimeric guanosine monophosphate (c-di-GMP) governs adaptive responses in the opportunistic pathogen Pseudomonas aeruginosa, including biofilm formation and the transition from acute to chronic infections. Understanding the intricate c-di-GMP signalling network remains challenging due to the overlapping activities of numerous diguanylate cyclases (DGCs). In this study, we employed a CRISPR-based multiplex genome-editing tool to disrupt all 32 GGDEF domain-containing proteins (GCPs) implicated in c-di-GMP signalling in P. aeruginosa PA14. Phenotypic and physiological analyses revealed that the resulting mutant was unable to form biofilms and had attenuated virulence. Residual c-di-GMP levels were still detected despite the extensive GCP disruption, underscoring the robustness of this regulatory network. Taken together, these findings provide insights into the complex c-di-GMP metabolism and showcase the importance of functional overlapping in bacterial signalling. Moreover, our approach overcomes the native redundancy in c-di-GMP synthesis, providing a framework to dissect individual DGC functions and paving the way for targeted strategies to address bacterial adaptation and pathogenesis.