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

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