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Physiology and Mechanisms of Pyocyanin Reduction In PHYSIOLOGY AND MECHANISMS OF PYOCYANIN REDUCTION IN PSEUDOMONAS AERUGINOSA Thesis by Alexa Mari Price-Whelan In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2009 (Defended February 4, 2009) ii 2009 Alexa Mari Price-Whelan All Rights Reserved iii Acknowledgements Throughout the process of obtaining my PhD, I have been extremely fortunate to meet and work with passionate and inspiring people. I will do my best to acknowledge all of them here. Technically, my work toward a degree in microbiology started before I knew I would be going to graduate school, when I met Jeanne S. Poindexter. Dr. P defined my college learning experience and changed my view of the natural world forever. I am so grateful that she did. At Caltech, I rotated in Dianne Newman’s lab and took her microbial metabolic diversity course. Through my discussions with her during this time, a ball dropped and I realized I needed to spend the next five years of my life thinking about redox-active small molecules and electron transfer in microbial physiology. I am not being sarcastic. I think it was a combination of many things: her enthusiasm and communication style, her ability to think about the subject at the level of a contamination site and at the level of a single electron at the same time, and also her general coolness. Usually big decisions in my life take months of misery, but this was a no-brainer. I joined her lab and I have never looked back. Dianne, thank you for accepting me as your student and supporting me as a scientist and an individual. I would like to thank Jared Leadbetter for the inspiring example he sets for scientists young and old. I would also like to thank Jacqueline Barton and Elliot Meyerowitz for their helpful suggestions throughout my time at Caltech. iv The Newman lab is a unique place, full of people who are eager to learn and discuss science from all fields, and I feel almost spoiled to have had the opportunity to work there. Everyone I overlapped with has contributed to this degree, but here I will mention particular influences that come to mind. Thanks to: Doug Lies for being a great colleague and encouraging me to think in pathways. Jeff Gralnick and Arash Komeili for offering me sage advice. Andreas Kappler for organic chemistry training. Laura Croal, Tracy Teal, and Davin Malasarn for friendship and guidance. Mariu Hernandez for starting the phenazine project. Itzel Ramos-Solis and Paula Welander for enlightening feminist discussions. Nicky Caiazza, for his wackiness and no-fuss approach. Sky Rashby, for his unique laugh. He is deeply missed. Yun Wang, for tantalizing chemistry discussions. Julie Huang, for contributing her huge natural talent to my project. Alex Poulain, for reminding me that science is exciting. Dave Doughty, for reminding me how fun plants are. Lina Bird and Suzanne Kern, for being beautiful people. Ryan Hunter, for just being a good guy. v I consider myself incredibly fortunate to have been raised in a household where critical thinking, discussion, creativity, and curiosity were respected and encouraged. Thank you, Mom and Dad, for passing this value system on to me. I can not imagine a life without it. During my time as a graduate student I met another person who fully subscribes to this value system and we fell in love. Lars, thank you for being my labmate and my lifemate. You totally get me. Finally, I would like to thank the people who supported me through the most exciting and also the most trying times of my PhD: My grandmother, Nana; my brother, Adrian; and the “fridge girls,” Lana Chung, Dina, and Yonit. Thank you for being my family. vi Abstract The opportunistic pathogen Pseudomonas aeruginosa excretes phenazines, redox- active toxins that historically have been categorized as secondary metabolites. This thesis addresses the possibility that pyocyanin, the most notorious phenazine produced by P. aeruginosa, acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined through a comparison of the physiological status of wild-type cells to those of a mutant defective in phenazine production, using two different techniques. Quantification of the intracellular NADH and NAD+ pools revealed that a phenazine-null mutant maintained a more reduced intracellular redox state than the wild type; this is consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of metabolites from cultures grown in defined media showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways. While mechanisms for pyocyanin redox cycling had been explored in other organisms, the mechanisms whereby P. aeruginosa catalyzes these reactions were largely unknown. A genetic screen was conducted to identify loci that contribute to the ability of P. aeruginosa PA14 to reduce ferric citrate, an activity that is phenazine dependent. This approach led to the identification of two loci with roles in pyocyanin reduction: (1) gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA or the cytochrome c-containing subunit (FbcC) of cytochrome bc1 displayed growth defects vii that correlate with the timing of pyocyanin production in batch cultures. The ΔgpsA mutant appeared to be unable to regulate its intracellular redox state and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, the ΔfbcC mutant produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction. Pyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form highly structured, rugose colonies, while the wild type forms smooth colonies. Through experiments with this colony biofilm assay, we showed that the ΔgpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in maintenance of redox homeostasis in densely packed communities. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results suggest roles for reduced pyocyanin and nitrate in triggering smooth colony formation in P. aeruginosa, and imply that colony wrinkling is an adaptation to electron acceptor limitation. The work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa and elaborated our understanding of the mechanisms underlying maintenance of redox homeostasis in bacteria. In addition, it has uncovered new mechanisms that may be contributing to the persistence of P. aeruginosa during chronic infection. viii Table of Contents Acknowledgements ....................................................................................................... iii Abstract..........................................................................................................................vi Table of Contents .........................................................................................................viii List of Figures...............................................................................................................xii List of Tables ...............................................................................................................xiv Chapter 1.........................................................................................................................1 Introduction.................................................................................................................1 1.1. Motivation ........................................................................................................1 1.2. Overview ..........................................................................................................2 1.3. References ........................................................................................................5 Chapter 2.........................................................................................................................6 Background .................................................................................................................6 2.1. Abstract ............................................................................................................6 2.2. Introduction ......................................................................................................7 2.3. Occurrence of Phenazine Production...............................................................11 2.4. Roles in Eukaryotic Physiology and Pseudomonad Persistence .......................13 2.4.1. Phenazines in Infection ............................................................................13 2.4.2. Phenazines in Soil Ecosystems.................................................................15 2.5. Biosynthesis of Phenazine Derivatives............................................................16 2.6. Intercellular Signaling: A Regulatory Role for Phenazines..............................19 2.7. Other Physiological Roles...............................................................................21 2.7.1. Phenazines as
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