Consequences of redox-active phenazines on the physiology of the opportunistic pathogen Pseudomonas aeruginosa by Suzanne E. Kern B.A. Biochemistry The Colorado College (2004) Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY IN BIOLOGY AT THE ARCHNE MASSACHUSETTS INSTITUTE OF TECHNOLOGY ASSACHUSETTS INS E OF TECHNOLOGY JUNE 2013 MAY 0 2 2013 @ Suzanne E. Kern. All rights reserved. The author hereby grants to MIT -IBRARIES permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of A uthor ........... / . ....... ............ ..... ............... ................................... Department of Biology March 29, 2013 Certified by ................... ................... Dianne K. Newman Professor of Biology Thesis Supervisor Accepted by........................................................... Amy E.Keating Professor of Biology Chairman, Committee for Graduate Studies 2 Consequences of redox-active phenazines on the physiology of the opportunistic pathogen Pseudomonas aeruginosa by Suzanne E. Kern Submitted to the Department of Biology on March 29, 2013 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT Phenazines are redox-active small molecules produced by bacteria. Although phenazines have been studied extensively for their roles as toxins, how phenazines benefit producing organisms is still being uncovered. Pseudomonas aeruginosa is a phenazine- producing Gram-negative bacterium that inhabits soil and water, and can establish persistent infections in plants, animals, and humans. P. aeruginosa produces phenazines upon activation of its quorum-sensing system, which is involved in numerous physiological changes, including biofilm development. Phenazines have been proposed to aid catabolism of P. aeruginosaunder conditions like those found in biofilms-rich in nutrients but low in suitable respiratory oxidants, e.g., oxygen and nitrate-and phenazines are known to oxidize nicotinamide adenine dinucleotides (NAD(P)H) and thereby affect biofilm structure and development. The work in this thesis demonstrates that P. aeruginosa PA14 can survive under anoxic conditions in the presence of glucose and oxidized endogenous phenazines, including pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxylic acid. Exogenous oxidants such as methylene blue, paraquat, and 2,6-anthraquinone disulphonate, do not support anaerobic survival, suggesting that phenazine survival is an evolved trait that enhances the fitness of P. aeruginosa. Phenazines enable anaerobic survival with glucose but not succinate, and pyruvate fermentation is important for this process. Phenazine redox cycling yields higher levels of ATP, likely by facilitating the oxidation of glucose to pyruvate and acetate by recycling NAD(P)H to NAD(P)+. ATP hydrolysis through the FoF1 ATPase sustains a membrane potential, which is necessary for survival. Similar results were observed for both pyruvate and arginine fermentation. Common features across these survival conditions included NADH/NAD+ ratios less than 3, a polarized membrane, and higher ATP levels than those measured in conditions that do not sustain viability. To perform this work, robust methods for quantifying NADH/NAD+ and phenazines were developed and are described herein. The findings of this thesis represent an important step forward in our understanding of how phenazines physiologically benefit the organisms that produce them. Furthermore, they point us to a more general model of survival for the opportunistic pathogen P. aeruginosa. Thesis Supervisor: Dianne K. Newman Title: Professor of Biology and Geobiology, California Institute of Technology and Investigator, Howard Hughes Medical Institute 3 4 Table of Contents Title Page 1 Abstract 3 Table of Contents 5 Acknowledgements 7 Chapter 1: Introduction 9 Chapter 2: Background 13 Chapter 3: Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer 35 Chapter 4: Pseudomonas aeruginosa has multiple metabolic pathways that enable anaerobic survival and maintenance of the membrane potential 51 Chapter 5: Method for the Extraction and Measurement of NAD(P)* and NAD(P)H 85 Chapter 6: Method for Measurement of Phenazines in Bacterial Cultures 103 Chapter 7: Method for assaying anaerobic survival by phenazine redox cycling 117 Chapter 8: Conclusions and Future Directions 139 Appendix A: Effects of varying experimental conditions on P. aeruginosa PA14 anaerobic survival with phenazine redox cycling 147 Appendix B: Applications of an assay to measure pyocyanin reduction rates by mutants of Pseudomonas aeruginosa 163 5 6 Acknowledgements I would like to thank many people for their assistance and support throughout my time as a PhD student. First and foremost, I am grateful to Dianne for taking me on as a student and fostering in me a sense of fascination about the world of microbial physiology, and for making it possible for me to follow the lab's move to Caltech. Through my experiences and our conversations, I have grown as a scientist and as a person. I want to thank all of the wonderful people I have overlapped with during my time in the group, especially Lars, Alexa, Yun, Nora, Jessie, Maureen, Chia, Lina, Seb, and Nate for engaging in helpful discussions. At the end of my third year at MIT, I moved with the lab to Caltech, which has turned out to be a great place to conduct research and to call home. Thanks to the many people who have been integral to my sense of community here, in particular the Resident Associates, Becky, Jill, Janet, and the undergrads of Fleming House who keep life interesting and provide balance to my life. The support of key staff and administrators at both Caltech and MIT has made a world of difference to me. Thank you to Betsey, Taso, Dean Hunt, Tess, Kristy, Olga, Mieke, Portia, Dean Staton, Araceli, and Jill. I also appreciate all of the encouragement and interest from my family members-Kern, Merdinger, Kempes, and Poling. Chris is at the top of this list for always being there for me, ready to talk about science or anything else. Finally, a warm thanks to my thesis committee members, Graham Walker, Penny Chisholm, and Ned Ruby, for your guidance and helpful suggestions. And I gratefully acknowledge the funding that has made it possible for me to pursue the interesting story of my research: NSF Graduate Research Fellowship Program, NIH training grant, and HHMI. 7 8 Chapter 1 Introduction 9 Motivation Unlike animals, which can move and maintain homeostasis in the face of environmental variation, single-celled organisms such as bacteria must instead acclimate to physical and chemical changes, or die. The formation of multicellular assemblages of bacteria, also known as biofilms, is a common mechanism for enhancing fitness under stressful environmental conditions. In the case of microbial pathogens, we are interested in understanding what underpins their ability to grow or survive within particular conditions so that other scientists might be able to design new and effective antimicrobial treatments. Pseudomonasaeruginosa is an environmental bacterium and opportunistic human pathogen capable of establishing infections that are highly resistant to eradication by antibiotic therapies. P. aeruginosaforms biofilms under a variety of conditions, and produces redox- active compounds called phenazines upon reaching high cell densities. Due to elevated levels of respiration by P. aeruginosa close to the surface, cells near the center of biofilms often inhabit niches with little to no oxygen. In such a context, when a terminal oxidant is in very limited supply, it has been proposed that soluble redox-active mediators could relieve the buildup of electrons that might exist when cells transition from aerobic to anaerobic conditions (Hernandez & Newman, 2001; Price-Whelan et al, 2006). In this thesis, I present evidence of the physiological benefits of phenazines to P. aeruginosa, an organism that produces and secretes these compounds at levels in the micromolar range. Overview Chapter 2 introduces the topic of phenazines and their physiological impacts on the organisms that produce them, with a particular focus on pseudomonads. While phenazines have long been recognized for their antibiotic properties, more recently they have garnered recognition for their role in the physiological development of biofilms and in survival under conditions of terminal-oxidant limitation. Parts of Chapter 2 will be included in a review chapter regarding the physiological impacts of phenazines in the forthcoming book Microbial Phenazines: Use in Agriculture, Energy and Health, published by Springer and compiled by Sudhir Chincholkar and Linda Thomashow. 10 Chapter 3 documents the discovery that phenazine redox-cycling can enable the anaerobic survival of P. aeruginosa. Using well-mixed planktonic cultures in an oxygen-free atmosphere, we approximated the oxygen-depleted conditions of a typical biofilm, where the redox activity of phenazines has been proposed to alleviate the stress of terminal- oxidant limitation (Hernandez & Newman, 2001; Price-Whelan et al, 2006). This work was published in the Journal of Bacteriology: Wang Y, Kern SE & Newman DK (2010) Endogenous phenazine antibiotics promote anaerobic survival of Pseudomonas aeruginosa via extracellular electron transfer.