Author ORCID Identifier

https://orcid.org/my-orcid?orcid=0000-0001-9723-7677

Date of Award

2025

Document Type

Thesis (Ph.D.)

Department or Program

Microbiology and Immunology

First Advisor

Deborah A. Hogan

Abstract

Pseudomonas aeruginosa is a prominent opportunistic pathogen that is often found in microoxic environments like sites of infection and colony biofilms. P. aeruginosa can grow at oxygen (O2) at concentrations as little as 3 μM and also grows anaerobically by generating energy using nitrate as an alternate electron acceptor. An important way P. aeruginosa adapts to low O2 is through Anr, a transcription factor that upregulates many genes important for microoxic and anoxic fitness.

Specifically, Anr regulates mhr which encodes a hemerythrin protein (Mhr) that binds O2 with affinities relevant to microoxia and is also necessary for full fitness of P. aeruginosa in colony biofilms. In this thesis, we present data that suggest Mhr is important for growth in colony biofilms, but not in planktonic cultures. Mhr has an 11-amino acid C- terminal extension not found in all hemerythrins that we show is necessary for membrane localization which is required for the Mhr contribution to fitness. Interestingly, Mhr has an epistatic relationship with the membrane-bound, high-affinity cbb3 oxidases that allow P. aeruginosa to respire O2 at low concentrations. Data presented in this thesis show that the presence of the cbb3 oxidases was also important for Mhr membrane localization. Together, these data suggest that the cbb3 oxidases promote Mhr membrane localization and contribute to the Mhr-dependent microoxic fitness phenotype.

Anr and the transcriptional regulator Dnr, also regulate denitrification, a process that allows P. aeruginosa to utilize nitrate as an alternative electron acceptor and ultimately transforms nitrate to nitrogen gas using a series of membrane-bound reductase enzymes. While denitrification is best described in the context of anaerobic growth, P. aeruginosa also performs denitrification in oxic conditions as well. The data in this thesis expand upon the contribution of oxic denitrification to P. aeruginosa fitness in commonly-used laboratory media like lysogeny broth (LB) and artificial sputum media. Our data show that Dnr and Dnr-regulated norB are important for P. aeruginosa fitness only in conditions where nitrate was being consumed. The work presented in this thesis presents important information about Anr-regulated factors that contribute to P. aeruginosa fitness in microoxic environments.

Original Citation

1. Stuut Balsam S, Zhong F, Pence N, Levintov L, Andhare D, Hammond JH, Ragusa MJ, Vashisth H, Hogan DA, Pletneva EV. Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin. Biochemistry. 2024 Jul 16;63(14):1795-1807. doi: 10.1021/acs.biochem.4c00174. Epub 2024 Jul 1. PMID: 38951132; PMCID: PMC11481101.

2. Role of Pseudomonas aeruginosa Dnr-regulated denitrification in oxic conditions Stacie Stuut Balsam, Dallas L. Mould, Fabrice Jean-Pierre, Deborah A. Hogan bioRxiv 2025.03.31.646406; doi: https://doi.org/10.1101/2025.03.31.646406

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