ABSTRACTThe opportunistic human pathogenPseudomonas aeruginosacauses chronic infections that involve multicellular aggregates called biofilms. Biofilm formation is modulated by the host environment and the presence of cues and/or signals, likely affecting the pool of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). The manganese ion Mn2+is a divalent metal cation that is essential for pathogenic bacterial survival and replication during the infection in a host organism. In this study, we investigated how Mn2+altersP. aeruginosabiofilm formation via the regulation of c-di-GMP levels. Exposure to Mn2+was found to temporally enhance attachment but impair subsequent biofilm development, apparent by reduced biofilm biomass accumulation and lack of microcolony formation due to the induction of dispersion. Moreover, exposure to Mn2+coincided with reduced production of the exopolysaccharides Psl and Pel, decreased transcriptional abundance ofpelandpsl, and decreased levels of c-di-GMP. To determine whether the effect of Mn2+was linked to the activation of phosphodiesterases (PDEs), we screened several PDE mutants for Mn2+-dependent phenotypes (attachment and polysaccharide production) as well as PDE activity. The screen revealed that the PDE RbdA is activated by Mn2+and is responsible for Mn2+-dependent attachment, inhibition of Psl production, and dispersion. Taken together, our findings suggest Mn2+is an environmental inhibitor ofP. aeruginosabiofilm development that acts through the PDE RbdA to modulate c-di-GMP levels, thereby impeding polysaccharide production and biofilm formation but enhancing dispersion.IMPORTANCEWhile diverse environmental conditions such as the availability of metal ions have been shown to affect biofilm development, little is known about the mechanism. Here, we demonstrate that Mn2+affectsPseudomonas aeruginosabiofilm development by stimulating phosphodiesterase RbdA activity to reduce the signaling molecule c-di-GMP levels, thereby hindering polysaccharide production and biofilm formation but enhancing dispersion. Our findings demonstrate that Mn2+acts as an environmental inhibitor ofP. aeruginosabiofilms, further suggesting manganese to be a promising new antibiofilm factor.