Optical O₂ Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria

From a metabolic perspective, molecular oxygen (O₂) is arguably the most significant constituent of Earth's atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O₂, which is the most favorable terminal electron acceptor used by organisms and also a dangerously reactive oxidant. As O₂ has such sweeping implications for physiology, researchers have developed diverse approaches to measure O₂ concentrations in natural and laboratory settings. Recent improvements to phosphorescent O₂ sensors piqued our interest due to the promise of optical measurement of spatiotemporal O₂ dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sensors, complicating O₂ 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 O₂ probe substantially. Phosphorescent O₂ probes are often embedded in solid support for protection, but an embedded probe called O₂NS 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 O₂NS. These findings underscore that optical O₂ measurements made in the presence of bacteria should be carefully controlled to ensure that O₂, and not bacterial secretions, is measured, and motivate the design of custom O₂ probes for specific organisms to circumvent sensitivity to redox-active metabolites. IMPORTANCE When they are closely packed, as in biofilms, colonies, and soils, microbes can consume O₂ faster than it diffuses. As such, O₂ concentrations in natural environments can vary greatly over time and space, even on the micrometer scale. Wetting soil, for example, slows O₂ diffusion higher in the soil column, which, in concert with microbial respiration, greatly diminishes [O₂] at depth. Given that variation in [O₂] has outsized implications for microbial physiology, there is great interest in measuring the dynamics of [O₂] in microbial cultures and biofilms. We demonstrate that certain classes of bacterial metabolites frustrate optical measurement of [O₂] with phosphorescent sensors, but also that some species (e.g., E. coli) do not produce problematic secretions under the conditions tested. Our work therefore offers a strategy for identifying organisms and culture conditions in which optical quantification of spatiotemporal [O₂] dynamics with current sensors is feasible.