The Diguanylate Cyclase YfiN of Pseudomonas aeruginosa Regulates Biofilm Maintenance in Response to Peroxide

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found extracellular polysaccharide (EPS) production, regulation of motility and induction of stress response pathways as contributing to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that, compared to the wild type (WT), static biofilms of the {Delta}yfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H2O2); this defect can be rescued through genetic complementation. Additionally, we found that the {Delta}yfiN mutant biofilms produce less c-di-GMP than WT, and that H2O2 treatment enhanced motility of surface-associated bacteria and increased cell death for the {Delta}yfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe. ImportanceWe build on previous findings that suggest that P. aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility and to insure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.