Fine tuning cyclic-di-GMP signaling in Pseudomonas aeruginosa using the type 4 pili alignment complex

To initiate biofilm formation it is critical for bacteria to sense a surface and respond precisely. Type 4 pili (T4P) have been shown to be important in surface sensing, however, mechanism(s) driving downstream changes important for the switch to biofilm growth have not been clearly defined. Here, using macroscopic bulk assays and single cell tracking analyses of Pseudomonas aeruginosa, we uncover a new role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two hybrid and bimolecular fluorescence complementation assays show that PilO physically interacts with SadC and that the PilO-SadC interaction inhibits SadCs activity resulting in decreased biofilm formation and increased motility. We show that disrupting the PilO-SadC interaction contributes to greater variation of cyclic-di-GMP levels among cells, thereby increasing cell-to-cell heterogeneity in the levels of this signal. Thus, this work shows that P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this nucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.