Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter
Molly Hydorn, Sathya Narayanan Nagarajan, Elizabeth Fones, Caroline S. Harwood, Jonathan Dworkin
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.
Introduction
The adaptive response of bacteria to environmental changes can be relatively specific, as in the case of two-component signaling that involves a single transcription factor regulating a limited number of genes. Alternatively, an adaptive response can be broad, such as the widespread physiological changes often referred to as the “stringent response” mediated by the nucleotides guanosine tetraphosphate (ppGpp) and pentaphosphate (pppGpp), collectively (p)ppGpp.
Materials and method
Strain construction
Strains were derived from B. subtilis 168 trpC2 except as noted and are listed in S1 Table. Strains were constructed by transformation using conventional methodology and where necessary, media was supplemented with either 100 µg/mL spectinomycin, 10 µg/mL kanamycin, 5 µg/mL chloramphenicol, or 1X MLS. Reporters at sacA were constructed using pSac-cm [76] derived plasmids (S2 Table), confirmed via whole plasmid sequencing via Plasmidsaurus or Genewiz, and sacA integration was confirmed by assaying growth on TSS/glc and nongrowth on TSS sucrose plates.
Results
We placed a sequence corresponding to the (p)ppGpp-sensitive riboswitch from the promoter of Desulfitobacterium hafniense ilvE [28] between an inducible promoter (Phyperspank) and the luc gene encoding firefly luciferase (luciferin 4-monooxygenase) (RsFluc; Figs 1A and S1). This construct was integrated at the sacA locus and luminescence measurements were performed during growth in S7/glucose defined medium in a microplate reader. Raw luminescence readings were normalized to OD600 (RLU/OD) (Fig 1B, blue).
Discussion
Here we develop and evaluate RsFluc, a novel dynamic riboswitch-based reporter of (p)ppGpp metabolism in B. subtilis. We confirm that RsFluc accurately reflects (p)ppGpp abundance using a variety of approaches, including mutagenesis of both reporter and host. In addition, known stringent response inducers such as inhibition of tRNA aminoacylation or a nutrient downshift robustly stimulate RsFluc activity.
Acknowledgments
We acknowledge the contributions of Abigail Whalen to the initial development of the riboswitch reporter and advice from other members of our laboratory. We thank Dale Whittington (UW Mass Spectrometry Center) for assistance with the HPLC-MS analysis. We thank Jonathan Jagodnik for helpful discussions about nucleotide-specific riboswitches and Frederico Gueiros Fihlo for comments on the manuscript.
Citation: Hydorn M, Nagarajan SN, Fones E, Harwood CS, Dworkin J (2025) Analysis of (p)ppGpp metabolism and signaling using a dynamic luminescent reporter. PLoS Genet 21(8): e1011691. https://doi.org/10.1371/journal.pgen.1011691
Editor: Jue D. Wang, University of Wisconsin-Madison, UNITED STATES OF AMERICA
Received: April 15, 2025; Accepted: August 11, 2025; Published: August 22, 2025
Copyright: © 2025 Hydorn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All Data are in the manuscript and/or Supporting information files
Funding: This work was supported by National Institutes of Health (https://www.nih.gov/) grant R35GM141953 to JD and United States Army Research Office (https://arl.devcom.army.mil/who-we-are/aro/) contract W911NF2110015 to CSH. MH acknowledges support from the Columbia University Graduate Training Program in Microbiology and Immunology (National Institutes of Health Allergy And Infectious Disease grant T32AI106711) and the Training in Cardiovascular Translational Research Training Grant (National Institutes of Health, Heart Lung and Blood Institute grant 5T32HL120826). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
