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Published December 20, 2022 | v2
Journal Article Open

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

  • 1. ROR icon California Institute of Technology

Abstract

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.

Additional Information

Thanks to Chelsey VanDrisse for supplying pyocyanin, Andrew Babbin for OXNANO beads, Lucas Meirelles and John Ciemniecki for assistance with toxoflavin and B. glumae cultivation. Thanks to Josh Goldford, Darcy McRose, Georgia Squyers, Lev Tsypin, and Shuangning Xu for useful conversations. This investigation was aided by a Postdoctoral Fellowship from The Jane Coffin Childs Memorial Fund for Medical Research (to A.I.F.) and NIH grants (1R01AI127850-01A1 and 1R01HL152190-01) to D.K.N. as well as the US Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research Bioimaging Science Program under subcontract B643823 (to K.C.) and the LLNL 3DQ Microscope Project, SCW1713.

Copyright and License

© 2022 Flamholz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Additional details

Created:
October 24, 2023
Modified:
January 9, 2024