PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. A defining aspect of P. aeruginosa is its ability to make pyocyanin, a colorful redox-active pigment that contributes to biofilm development and its fitness in the context of infection. Pyocyanin has been detected at appreciable concentrations in skin wounds and in cystic fibrosis sputum, and pyocyanin has been shown to be a virulence factor in animal infection models. Pyocyanin exerts a range of effects over the cells that produce it, ranging from toxic in the presence of oxygen to beneficial in its absence; under oxygen-limited conditions, pyocyanin serves as an electron acceptor that promotes redox-balancing and long-term survival. These toxic and beneficial roles are important at different times in biofilm development, with early pyocyanin -promoted lysis generating extracellular DNA (eDNA), a key component of the biofilm matrix together with exopolysaccharides. Recently, we determined that eDNA underpins pyocyanin’s ability to promote extracellular electron transfer (EET) within biofilms, facilitating metabolic activity in the oxygen-limited interior. We found that eDNA enables both the retention of pyocyanin and charge transfer to pyocyanin. Critical to making these discoveries was our development of new bioelectrochemical technologies and approaches and the application of advanced spectroscopic techniques to directly probe EET in biofilms. We now seek to extend our interdisciplinary work to gain a mechanistic understanding of how biofilm EET efficiency is tuned by the composition of the matrix and the consequences this may have for antibiotic tolerance. Does the ratio of certain exopolysaccharides (Pel, Psl) to eDNA modulate pyocyanin diffusivity in the matrix, controlling EET efficiency? Does EET efficiency correlate with the rate of redox balancing in the biofilm interior? How do pyocyanin-mediated cellular effects, including EET, contribute to antibiotic tolerance in biofilms, and do these mechanisms differ according to the amount of oxygen in the microenvironment? Does the relative sensitivity of diverse pyocyanin-producing P. aeruginosa isolates to antibiotics correlate with their matrix composition and EET efficiency? Aim1 will explore how the matrix composition, particularly the ratio of Pel and Psl exopolysaccharides to eDNA, determines EET efficiency. Aim 2 will test the hypothesis that pyocyanin is a versatile intrinsic tolerance factor, where PYO-EET helps P. aeruginosa biofilms tolerate mechanistically distinct and clinically important antibiotic classes via bioenergetic effects and/or by inducing defense mechanisms; we predic...