Optical O(2) Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria

From a metabolic perspective, molecular oxygen (O(2)) is arguably the most significant constituent of Earth’s atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O(2), which is the most favorable terminal electron acceptor used by organisms and also a dangerously reactive oxidant. As O(2) has such sweeping implications for physiology, researchers have developed diverse approaches to measure O(2) concentrations in natural and laboratory settings. Recent improvements to phosphorescent O(2) sensors piqued our interest due to the promise of optical measurement of spatiotemporal O(2) dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sensors, complicating O(2) measurements in PA14 cultures and biofilms. Assaying supernatants from cultures of 9 bacterial species demonstrated that this phenotype is common: all supernatants quenched a soluble O(2) probe substantially. Phosphorescent O(2) probes are often embedded in solid support for protection, but an embedded probe called O(2)NS was quenched by most supernatants as well. Measurements using pure compounds indicated that quenching is due to interactions with redox-active small molecules, including phenazines and flavins. Uncharged and weakly polar molecules like pyocyanin were especially potent quenchers of O(2)NS. These findings underscore that optical O(2) measurements made in the presence of bacteria should be carefully controlled to ensure that O(2), and not bacterial secretions, is measured, and motivate the design of custom O(2) probes for specific organisms to circumvent sensitivity to redox-active metabolites.