A c-di-GMP signaling module controls responses to iron in Pseudomonas aeruginosa. Iron regulates c-di-GMP levels in P. aeruginosa by modulating the interaction between an iron-sensing protein, IsmP, and a diguanylate cyclase, ImcA. Binding of iron to the CHASE4 domain of IsmP inhibits the IsmP-ImcA interaction, leading to increased c-di-GMP synthesis by ImcA, thus promoting biofilm formation and reducing bacterial motility. Structural characterization of the apo-CHASE4 domain and its binding to iron allows us to pinpoint residues defining its specificity. The cryo-electron microscopy structure of ImcA in complex with a c-di-GMP analog (GMPCPP) suggests a unique conformation in which the compound binds to the catalytic pockets and to the membrane-proximal side located at the cytoplasm. Our results indicate that a CHASE4 domain directly senses iron and modulates the crosstalk between c-di-GMP metabolic enzymes. In response to distinct environmental stresses, bacterial cell survival is facilitated by the formation of biofilms. Cyclic dimeric GMP (c-di-GMP), a second messenger in bacteria, is the predominant regulator of biofilm formation and motility. Previous research has demonstrated that bacterial motility is partially regulated by different concentrations of c-di-GMP. For instance, higher/lower concentrations of c-di-GMP promote sessility/motility, and c-di-GMP-mediated processes are related to infection and host-microbe symbiosis. The synthesis and degradation of c-di-GMP are mainly associated with two unique enzymes called diguanylate cyclase (DGC) and phosphodiesterase (PDE). Numerous studies have been reported that bacteria possess multiple c-di-GMP-related enzymes, and distinct phenotypes, including biofilm dispersal and motility are controlled by their corresponding DGCs and/or PDEs. For example, the local PdeR-DgcM-MlrA signaling module controls the expression of the biofilm regulator CsgD in E. coli. DgcC and PdeK mutual interaction controls bacterial cellulose synthase (Bcs). Notably, the interaction hubs involving a few DGCs and PDEs in E. coli NosP-NahK and Pseudomonas fluorescens mediate c-di-GMP responses. Recently, direct interactions among the bacterial DGCs and PDEs have attracted much attention from researchers because these enzymes may perform similar catalytic functions to realize their different cellular decisions. The enzymatic activity of DGCs and PDEs is activated or repressed under different environmental cues. These enzymes often contain various N-terminal signaling domains such as the HAMP domain, PAS/PAC domain, GAF domain, CACHE domain, and CHASE domain. Each N-terminal signaling domain is able to sense distinctA c-di-GMP signaling module controls responses to iron in Pseudomonas aeruginosa. Iron regulates c-di-GMP levels in P. aeruginosa by modulating the interaction between an iron-sensing protein, IsmP, and a diguanylate cyclase, ImcA. Binding of iron to the CHASE4 domain of IsmP inhibits the IsmP-ImcA interaction, leading to increased c-di-GMP synthesis by ImcA, thus promoting biofilm formation and reducing bacterial motility. Structural characterization of the apo-CHASE4 domain and its binding to iron allows us to pinpoint residues defining its specificity. The cryo-electron microscopy structure of ImcA in complex with a c-di-GMP analog (GMPCPP) suggests a unique conformation in which the compound binds to the catalytic pockets and to the membrane-proximal side located at the cytoplasm. Our results indicate that a CHASE4 domain directly senses iron and modulates the crosstalk between c-di-GMP metabolic enzymes. In response to distinct environmental stresses, bacterial cell survival is facilitated by the formation of biofilms. Cyclic dimeric GMP (c-di-GMP), a second messenger in bacteria, is the predominant regulator of biofilm formation and motility. Previous research has demonstrated that bacterial motility is partially regulated by different concentrations of c-di-GMP. For instance, higher/lower concentrations of c-di-GMP promote sessility/motility, and c-di-GMP-mediated processes are related to infection and host-microbe symbiosis. The synthesis and degradation of c-di-GMP are mainly associated with two unique enzymes called diguanylate cyclase (DGC) and phosphodiesterase (PDE). Numerous studies have been reported that bacteria possess multiple c-di-GMP-related enzymes, and distinct phenotypes, including biofilm dispersal and motility are controlled by their corresponding DGCs and/or PDEs. For example, the local PdeR-DgcM-MlrA signaling module controls the expression of the biofilm regulator CsgD in E. coli. DgcC and PdeK mutual interaction controls bacterial cellulose synthase (Bcs). Notably, the interaction hubs involving a few DGCs and PDEs in E. coli NosP-NahK and Pseudomonas fluorescens mediate c-di-GMP responses. Recently, direct interactions among the bacterial DGCs and PDEs have attracted much attention from researchers because these enzymes may perform similar catalytic functions to realize their different cellular decisions. The enzymatic activity of DGCs and PDEs is activated or repressed under different environmental cues. These enzymes often contain various N-terminal signaling domains such as the HAMP domain, PAS/PAC domain, GAF domain, CACHE domain, and CHASE domain. Each N-terminal signaling domain is able to sense distinct