All ETDs from UAB

Advisory Committee Chair

Michael J Gray

Advisory Committee Members

Peter Prevelige

Todd Green

Natalia Kedishvili

Janet Yother

Document Type

Dissertation

Date of Award

2023

Abstract

Polyphosphate (polyP) is an ancient, conserved, inorganic biomolecule. Biological systems have adapted many functions for this high-energy molecule, ranging from immune regulation in mammals to gene regulation in prokaryotes. Of particular interest is its use in bacterial stress responses. Bacteria use polyP to resist hazardous environmental elements like toxic molecules or nutrient starvation. In many bacterial species, polyP is synthesized by polyphosphate kinases (PPKs). PPK – discovered in Escherica coli (E. coli) – hydrolyzes adenosine-5’-triphosphate (ATP) to synthesize polyP. Species that use PPK develop multiple pathogenic defects when PPK activity is impaired, including, among others, antibiotic susceptibility. There is no mammalian homolog of PPK, making it a prime therapeutic target. Unfortunately, there are many unanswered questions regarding PPK and polyP synthesis regulation. In this work we report several novel PPK regulatory elements. We begin with a review of the state of polyP research and an exploration of the complicated interaction of bacterial and host polyP utilization. Next, we identify new regulatory elements of polyP synthesis using genetic techniques. Nitrogen-regulation elements such as the nitrogen phosphotransferase regulator PtsN and nitrogen metabolism factors GlnG and GlrR were discovered to have regulatory effects on polyP production. Cellular nitrogen levels were implicated in RpoN-dependent polyP synthesis, though multiple factors in nitrogen regulation were eliminated as potential as PPK regulators. We next characterize purified PPK enzymes. Our results indicate that the widely used polyhistidine tag significantly alters PPK activity, oligomeric stability, and sensitivity to substrate inhibition. We describe an alternative purification method using the C-tag system and confirm its similarity to untagged enzyme. The therapeutic PPK inhibitor mesalamine (5-aminosalicylic acid, 5-ASA), a poor inhibitor of polyH-tagged PPK, was found to be more effective when tested on the C-tagged enzyme. Finally, subtle differences in the previously described PPK* mutant enzymes were identified that suggest that each mutation leads to increased polyP accumulation through different mechanisms. We conclude with data regarding the development of PPK purification and detection methods and propose several questions to inform future polyP studies. Collectively, this project has identified multiple novel regulatory elements in polyP biosynthesis which will inform future studies of polyP biology.

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