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

ABSTRACT 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. Multi-domain 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. IMPORTANCE The results presented in this study reveal a fascinating versatility of the CHASE4 domain in PA14 Pseudomonas aeruginosa cyclic di-GMP signaling systems (i.e., PA14_53310 and PA14_37690), highlighting its ability to sense different environmental cues, such as heme and copper. In the context of infection, the interplay between heme and copper sensing could be particularly relevant, being both nutrients or poisons. Their sensing (and adaptive response) would be a significant advantage for the bacterium, allowing it to optimize its virulence and survival strategies. Therefore, these results represent the starting point for future in vivo studies aimed at exploring the relevance of heme and copper in pathogenesis, to finally develop novel anti-virulence strategies targeting the pathogen’s ability to adapt to the host environment.