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Physiological and Biochemical Mechanisms of Phenazine-Mediated Survival in Pseudomonas aeruginosa

Citation

Glasser, Nathaniel Robert (2017) Physiological and Biochemical Mechanisms of Phenazine-Mediated Survival in Pseudomonas aeruginosa. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9SN070S. http://resolver.caltech.edu/CaltechTHESIS:05312017-133325449

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 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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Pseudomonas aeruginosa; phenazines; pyocyanin; redox homeostasis; metabolomics; crystallography; anaerobic
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Newman, Dianne K.
Thesis Committee:
  • Mazmanian, Sarkis K. (chair)
  • Rees, Douglas C.
  • Leadbetter, Jared R.
  • Newman, Dianne K.
Defense Date:26 April 2017
Non-Caltech Author Email:nateglasser (AT) gmail.com
Funders:
Funding AgencyGrant Number
NSF Graduate Research Fellowship1144469
Record Number:CaltechTHESIS:05312017-133325449
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05312017-133325449
DOI:10.7907/Z9SN070S
Related URLs:
URLURL TypeDescription
http://doi.org/10.1111/mmi.12566DOIArticle adapted for Chapter 3
http://doi.org/10.1074/jbc.M116.772848DOIArticle adapted for Chapter 4
http://doi.org/10.1126/science.aag3180DOIArticle adapted for Chapter 6
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10230
Collection:CaltechTHESIS
Deposited By: Nathaniel Glasser
Deposited On:09 Jun 2017 21:09
Last Modified:16 Jun 2017 22:33

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