2025 Theses Doctoral

A comprehensive analysis of (p)ppGpp signaling in bacteria

Hydorn, Molly Anne

(p)ppGpp is a signaling molecule produced by bacteria in response to stresses, particularly amino acid starvation. RelA/SpoT homolog (RSH) enzymes synthesize (p)ppGpp when an uncharged tRNA enters an actively translating ribosome, stalling protein synthesis. (p)ppGpp slows bacterial growth by inhibiting translation, transcription, and DNA replication, and it upregulates the transcription of stress response genes. (p)ppGpp has also been shown to increase pathogenicity, virulence, and antibiotic tolerance.

For example, persister cells — cells that are tolerant to antibiotic exposure and result in persistent and chronic infections — are thought to arise from a subpopulation of cells that are high in (p)ppGpp. Current methods of detecting (p)ppGpp are limited to bulk measurements using inaccessible techniques, making analysis of (p)ppGpp metabolism and its physiological consequences challenging. Here, I develop RsFluc, a novel tool for (p)ppGpp detection in B. subtilis, and validate it using HPLC-MS and known (p)ppGpp induction methods. I use RsFluc to assess (p)ppGpp metabolism in B. subtilis and assay the environmental stimuli that trigger (p)ppGpp signaling.

I evaluate the downstream responses to (p)ppGpp using RsFluc, transcriptional reporters, and protein synthesis labeling techniques. I develop RsGFP, a tool for (p)ppGpp detection at the single-cell level. Using RsGFP, I evaluate heterogeneity in (p)ppGpp throughout growth and the factors that contribute to that heterogeneity. I utilize these (p)ppGpp detection tools to develop a novel single-cell RNA sequencing (scRNA-seq) technique that enables researchers to assay heterogeneity in (p)ppGpp while simultaneously assessing differential gene expression at the single-cell level. I use RsGFP to evaluate the relationship between heterogeneity in (p)ppGpp and heterogeneity in regrowth and antibiotic tolerance.

Lastly, collaborators and I developed a technique for labeling cells for protein synthesis while performing scRNA-seq, gaining insights into physiological states and how these states may affect transcription.