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bioRxiv · 10.1101/2025.04.16.649062

Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter

Hydorn, M.·Nagarajan, S. N.·Fones, E.·Harwood, C.·Dworkin, J.
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Abstract

As rapidly growing bacteria begin to exhaust nutrients, their growth rate slows, ultimately leading to the non-replicative state of quiescence. Adaptation to nutrient limitation requires widespread metabolic remodeling that is in part mediated by the phosphorylated nucleotides guanosine tetra- and penta-phosphate, collectively (p)ppGpp. We have developed a novel reporter of (p)ppGpp abundance in the Gram-positive bacterium Bacillus subtilis based on the recent identification of a riboswitch that binds (p)ppGpp and modulates transcription via regulation of a transcriptional terminator. Placement of an unstable reporter, firefly luciferase, downstream of the riboswitch allows for sensitive and dynamic assessment of (p)ppGpp. We first confirm that the reporter accurately reflects (p)ppGpp abundance in a variety of well-established conditions. We then proceed to use it to demonstrate the physiological importance of several mechanisms of regulation of (p)ppGpp metabolism previously observed only in vitro including allosteric interactions between (p)ppGpp synthesis enzymes and the hydrolytic activity of a (p)ppGpp synthase. (p)ppGpp signaling has been implicated in the regulation of gene expression, and we demonstrate a close temporal association between gene expression and (p)ppGpp abundance, indicating a rapid, and therefore likely direct mechanism of (p)ppGpp dependent gene activation. Thus, this reporter provides a new, comprehensive analysis of (p)ppGpp signaling in vivo and offers the potential ability to sensitively monitor the temporal dynamics of (p)ppGpp abundance under diverse environmental conditions. Author SummaryMost bacteria adapt to stressful conditions such as nutrient limitation by synthesizing a signaling molecule, known as ppGpp, that consists of a hyper-phosphorylated GTP. Synthesis of ppGpp affects most aspects of cellular physiology including replication, transcription and translation. We present here a method that for the first time allows measurement of ppGpp abundance in living cells, greatly facilitating investigation into ppGpp metabolism.

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Hydorn, M., Nagarajan, S. N., Fones, E., Harwood, C., Dworkin, J.. 2025-04-16. Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter. https://doi.org/10.1101/2025.04.16.649062

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