The versatility of the CHASE domain in one-component systems of c-di-GMP signal transduction from P. aeruginosa

Cyclic di-GMP (c-di-GMP) is a key bacterial second messenger that regulates a wide range of cellular processes, including biofilm formation and virulence. Multidomain one-component systems regulate c-di-GMP synthesis and turnover in response to external signals. Periplasmic sensing of environmental cues is performed by versatile but conserved sensory domains, including members of the CHASE4 superfamily. Here, we explore the promiscuity of the CHASE4 domain in c-di-GMP signal transduction in Pseudomonas aeruginosa by analyzing two CHASE4-containing transducers from the virulent PA14 strain, namely PA14_53310 and PA14_37690. With the integration of biochemical and biophysical methods, such as UV-Vis spectroscopy, circular dichroism, and isothermal titration calorimetry, we demonstrate that the CHASE4 domains of these proteins possess different ligand specificities. We show that PA14_53310 is a diguanylate cyclase, and that its enzyme activity is controlled by heme binding to its periplasmic CHASE4 domain. The PA14_37690 CHASE4 domain, on the other hand, does not bind heme, but likely recognizes copper, indicating a role in metal ion sensing. A comparison with the PAO1 counterpart, that is, PA0847 and PA2072, is discussed and the divergences highlighted. This work demonstrates that the CHASE4 domain is a multifunctional sensory module that can be calibrated to detect a range of environmental cues, thus providing a mechanism for c-di-GMP signaling refinement in P. aeruginosa. These results shed light on the molecular basis for the functional diversification of CHASE4-containing one-component systems and their role in bacterial adaptation.