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dc.contributor.authorWilson, Tyree
dc.date.accessioned2022-09-14T15:21:57Z
dc.date.available2022-09-14T15:21:57Z
dc.date.issued2022
dc.identifier.urihttp://hdl.handle.net/10713/19799
dc.descriptionUniversity of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 2022en_US
dc.description.abstractPseudomonas aeruginosa is an opportunistic pathogen that requires iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either system is eventually sequestered by the cytoplasmic protein PhuS, which specifically shuttles heme to the iron-regulated heme oxygenase HemO. Furthermore, a conformational rearrangement upon heme binding is necessary for the protein-protein interaction with HemO and a ligand switch between the heme coordinating ligands (His209 and His212) was proposed ot be required for translocation of heme to HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron- responsive sRNAs prrF1, F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, can also modulate PrrF and PrrH levels in response to heme. The dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host. Additional biophysical, genetic and metabolic approaches have been conducted to determine the role of the PhuS heme coordinating residues regulate the mutual exclusivity of heme and DNA binding and the resulting effects on PrrF and PrrH expression.en_US
dc.language.isoen_USen_US
dc.subjectprotein-DNA interactionsen_US
dc.subjectregulatory sRNAsen_US
dc.subjectconformational dynamicsen_US
dc.subject.meshHemeen_US
dc.subject.meshPseudomonas aeruginosaen_US
dc.titleThe Regulatory Role of the Cytoplasmic Heme Binding Protein PhuS in Pseudomonas aeruginosaen_US
dc.typedissertationen_US
dc.date.updated2022-09-06T19:12:26Z
dc.language.rfc3066en
dc.contributor.advisorWilks, Angela
refterms.dateFOA2022-09-14T15:21:58Z


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