A Physicochemical Approach to Determining the Functions of Microbial Phenazine Metabolites

Author: Thalhammer, Korbinian O.

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Newman, Dianne K.

Committee Members: Eiler, John M.; Sessions, Alex L.; Leadbetter, Jared R.; Newman, Dianne K.

Option: Geochemistry

Abstract

Phenazines are redox-active microbial metabolites produced in diverse ecologic niches from agricultural soils to chronic infections. Hundreds of phenazine analogs are known to exist in nature, but their precise roles in context and the reasons for their chemical diversity remain elusive. While much energy has been devoted to investigating phenazine biology, laboratory experiments designed around phenotypes often neglect important aspects of the environmental conditions in which phenazines function, namely pH and EH. In this thesis, I propose that an alternative route to determining the true evolved functions of phenazines and other secondary metabolites is to first interrogate their physicochemical properties in relevant context and to then allow the results to guide biological questions. As a case study, I made detailed abiotic lipophilicity measurements of the phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa. The measurements revealed an elegant redox-mediated mechanism by which lipophilicity is tuned in vivo, sometimes by several orders of magnitude. The increase in biologic retention implied by this finding was born out experimentally by the discovery that the P. aeruginosa membrane harbors millimolar concentrations of reduced pyocyanin in low oxygen conditions, a finding that upends the prevailing concept of that metabolite as an extracellular toxin actively secreted by producer cells. This finding in turn raised questions about the integrity of the pyocyanin-saturated membrane and the function of respiratory enzymes therein. The first question inspired a preliminary lipidomics study that points to significant lipid remodeling in the presence of phenazines. The second may be addressed in the future by respirometry methods developed here for interrogating phenazine interactions with the P. aeruginosa electron transport chain. In the final chapter, I describe attempts to address whether phenazine-mediated reduction of Fe(III)-bearing clays in the environment is a viable mechanism of anaerobic survival for environmental phenazine producers. The experiments described throughout this work represent a fundamentally chemical approach to biological questions, and the results speak to the value of that perspective.