Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa

: The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.