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Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oxidative DNA damage can lead to cancer, and as enzymatic DNA repair systems become compromised during the aging process, the role of exogenous antioxidants becomes more critical. Here, we examined whether such non-enzymatic DNA repair can be effected by the common cellular antioxidant glutathione, investigating both permanent DNA damage products and the guanine radical intermediates that form them, using the flash-quench technique to carry out the one-electron oxidation of guanine. In gel-shift assays, the presence of reduced glutathione at physiological (millimolar) concentrations strongly inhibits oxidative DNA-protein cross-linking. In contrast, the oxidized glutathione dimer affords only a minimal amount of protection, even at elevated pH where there is more of the strongly reducing thiolate form. In flash photolysis experiments, the formation and decay of the guanine neutral radical were monitored at 510 nm. Transient absorption measurements with a guanine-rich 22-mer DNA duplex on the millisecond time scale show that the yield of this long-lived signal is significantly diminished in the presence of reduced glutathione, suggesting a reduction process that is fast relative to the measurement. Indeed, transient absorption experiments carried out on faster time scales show that the microsecond decay of the guanine radical signal is visibly faster with glutathione present. Glutathione is perhaps best known as an electron source in enzymatic reactions, to maintain cysteines in reduced states in proteins and to deactivate reactive oxygen species. However, these results show that another important task for glutathione may be to directly intercept DNA radicals before permanent DNA damage can occur.