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Physiological and Biochemical Mechanisms of Phenazine- Mediated Survival in Pseudomonas aeruginosa Thesis by Nathaniel Robert Glasser In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2017 (Defended April 26, 2017) ii © 2017 Nathaniel Robert Glasser All Rights Reserved iii ACKNOWLEDGEMENTS I owe any accomplishments to my family: to my father, for teaching me a love of math and science; to my mother, for teaching me a love of reading and writing; and to my sister, for grounding me with music and fun. None of my research would be possible without the wonderful group of people Dianne has assembled. To Dianne and all the group members I’ve had the pleasure of working with: Brittany, Caj, Chia, Christian, Dave, David, Elena, Elise, other Elise, Flavia, Gargi, Jessica, Kristy, Kyle, Lina, Lindsey, Lisa, Lucas, Maureen, Megan, Melanie, Michael, Naomi, Nick, Olga, Peter, Ruth, Ryan, Scott, Shannon, Seb, Tahoura, and Will. A special recognition for my co-authors and collaborators: Suzanne, for never giving up; Ben, for your relentless enthusiasm; Julie, for your supernatural calmness; Kyle, for building excellent settlements; and Stuart, for teaching us biologists where to push the electrons. To my human friends, Marissa, Josh, and Paul, and my furry friends, Archimedes and Hypatia, for making game night the highlight of my week; and to Natalie Martin, for bringing me food late at night. And to those with whom I’ve shared invaluable scientific discussions: Ned Ruby, Ian Booth, Nathan Dalleska, Jens Kaiser, Shu-ou Shan, and Andre Hoelz; and finally to my committee, Doug Rees, Jared Leadbetter, and Sarkis Mazmanian. Thank you all! iv ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa secretes a class of colorful redox-active small molecules known as phenazines. Numerous functions have been proposed for phenazines, including antibiotic activity, virulence, cell-to-cell signaling, iron acquisition, and survival. This thesis delves into mechanisms of the latter role, that of long-term survival under oxidant-limiting conditions. Using a diverse array of methods, I investigated how phenazines support survival and how cells transfer electrons to phenazines, as well as the downstream effects that phenazines have on P. aeruginosa. Direct measurements of NAD(H), ATP, the membrane potential, and fermentation products revealed that phenazines promote redox homeostasis and subsequently ATP synthesis. The ATP is used to maintain a membrane potential through the reverse action of the ATP synthase complex. Even though P. aeruginosa does not ferment on sugars, phenazines enable the anaerobic oxidation of glucose to acetate, suggesting P. aeruginosa may have previously under-appreciated metabolic flexibility in the absence of terminal electron acceptors. Activity assays with proteins purified natively from P. aeruginosa showed that glucose oxidation might be enabled in vivo by the pyruvate dehydrogenase complex, which can directly reduce phenazines using pyruvate as an electron donor. Liquid chromatography and mass spectrometry of culture supernatants showed that phenazines alter the chain length distribution of secreted quinolones, which may have indirect downstream signaling effects. Based on this result, combined with data from survival experiments, I hypothesize that phenazine-mediated redox homeostasis promotes β-oxidation and that fatty acid metabolism contributes to long-term survival. Further analysis also showed that P. aeruginosa cultures contain several previously-unreported sulfonated phenazines. In its natural environment, P. aeruginosa undoubtedly encounters other microbial species that consume or modify its phenazines. At least one of these, a Mycobacterium, contains a pyocyanin demethylating v enzyme. The X-ray crystal structure of this protein revealed a novel reaction mechanism wherein the substrate is its own electron acceptor. Together, this work illuminates some of the many ways phenazines shape microbial communities in both clinical and environmental contexts. vi PUBLISHED CONTENT AND CONTRIBUTIONS Glasser, N. R., Saunders, S., and Newman, D. K. (2017) The colorful world of extracellular electron shuttles.Annual Review of Microbiology. In Press. N.R.G. participated in collecting and reviewing the literature, created the figures, and wrote the sections discussing the chemistry and biochemistry of electron shuttling. Glasser, N. R., Kern, S. E., and Newman, D. K. (2014) Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton- motive force. Molecular Microbiology 92, 399-412. DOI: 10.1111/mmi.12566 N.R.G. collected the data and participated in designing the experiments and writing the manuscript. Glasser, N. R., Wang, B. X., Hoy, J. A., and Newman, D. K. (2017) The pyruvate and α- ketoglutarate dehydrogenase complexes of Pseudomonas aeruginosa catalyze pyocyanin and phenazine-1-carboxylic acid reduction via the subunit dihydrolipoamide dehydrogenase. Journal of Biological Chemistry 292, 5593-5607. DOI: 10.1074/jbc.M116.772848 N.R.G. collected the data, refined the protein structures, wrote the manuscript, and helped design the experiments. Costa, K. C., Glasser, N. R., Conway, S. J., and Newman, D. K. (2017) Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms. Science 355 (6321), 170- 173. DOI: 10.1126/science.aag3180 N.R.G. assisted with protein crystallization and solving the X-ray crystal structure, and with writing the relevant methods sections of the manuscript. vii TABLE OF CONTENTS Chapter 1 1 Introduction .............................................................................................................................................. 1 Overview ............................................................................................................................................ 3 Literature Cited ................................................................................................................................ 5 Chapter 2 6 Background ............................................................................................................................................... 6 Abstract............................................................................................................................................... 6 Introduction ...................................................................................................................................... 6 Diversity of Endogenous EES and Organisms ........................................................................... 9 Chemical diversity of EES .................................................................................................... 11 Phylogenetic diversity ........................................................................................................... 12 Costs of EES Biosynthesis ........................................................................................................... 15 Cell Biology of Electron Shuttling ............................................................................................. 16 EES in the Extracellular Environment ....................................................................................... 20 Shuttle diffusion ...................................................................................................................... 20 Electron hopping as an alternative to shuttle diffusion .................................................. 22 Evolutionary strategies that maintain EES in open environments ............................... 22 Outlook ........................................................................................................................................... 24 Sidebars ........................................................................................................................................... 25 Sidebar 1. How to characterize a putative EES ................................................................ 25 Sidebar 2. How does redox-potential define an EES? ..................................................... 27 Glossary ........................................................................................................................................... 28 Acknowledgements ....................................................................................................................... 28 Literature Cited ............................................................................................................................. 28 Chapter 3 38 Physiology of Phenazine-Mediated Survival ................................................................................... 38 Abstract............................................................................................................................................ 38 Introduction ................................................................................................................................... 39 Results .............................................................................................................................................. 40 Phenazine redox cycling requires the activity of acetate kinase to promote survival 40 Phenazine redox cycling promotes ATP synthesis during pyruvate fermentation ... 45 ATP synthesis is limited by redox homeostasis during pyruvate fermentation ......... 47 Arginine supports anaerobic
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