# The critical roles of (p)ppGpp in homeostasis and antibiotic tolerance in Gram positive bacteria

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $456,560

## Abstract

Project Summary
 Bacteria frequently encounter stresses including nutrient starvation, temperature changes, and
antibiotic assault, which could easily throw their intracellular environment into chaos. To survive and
to adapt, bacteria developed diverse stress responses to regulate intracellular processes
accordingly. While the transcriptional networks governing stress responses have been extensively
characterized, there are major gaps in our knowledge beyond transcription regulation. The theme of
my research is to elucidate stress signaling mechanisms that are transmitted by rapid changes in
concentration of ‘alarmones’ – signaling nucleotides which are instrumental for alerting cells about
stresses in a timely manner. My laboratory has extensive experience in characterizing the conserved
alarmone (p)ppGpp. (p)ppGpp is induced by stresses and mediates profound, pleiotropic
physiological changes in almost all bacteria to allow fitness, survival, and evolution. We identified
multiple purine synthesis enzymes, a replication enzyme and a transcription repressor that are
directly regulated by (p)ppGpp in Gram-positive Bacillus species. These regulations were further
found to be conserved in many pathogens and are critical for homeostasis, starvation resistance,
antibiotic persistence, and genome integrity. Currently, we are also investigating how (p)ppGpp
regulates the switch between distinct bacterial lifestyles: planktonic growth and biofilm formation.
Additionally, we detected other nucleotide alarmones including AppppA, pGpp, ppApp, and c-di-
AMP, which are induced by different stresses including temperature and cell wall stress, to form a
robust protective network. Our future research will answer the following fundamental questions: How
are the different alarmones triggered by different stresses, and how do bacteria synthesize them?
What are the direct interaction targets of different alarmones, and how do they promote bacterial
fitness and influence bacterial development such as biofilm formation and sporulation? How do
bacteria integrate multiple cues from different alarmones for rapid and appropriate adaptation to
diverse environments? We combine metabolomics, transcriptomics, and proteomics with
biochemical and cell biological approaches to answer these questions. We obtained a list of
alarmone targets from systematic screens performed with the proteome of the pathogen Bacillus
anthracis. We will study these processes in the related non-pathogenic bacterium Bacillus subtilis
for which we have extensive experience. B. subtilis grows fast and is highly amenable to genetic
manipulation. The nucleotide signaling mechanisms we characterize in Bacillus are applicable to
other, less tractable, pathogenic bacteria, and can be used for developing antimicrobial strategies
by targeting their stress responses.

## Key facts

- **NIH application ID:** 10833623
- **Project number:** 5R35GM127088-07
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Jue D. Wang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $456,560
- **Award type:** 5
- **Project period:** 2018-05-01 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10833623

## Citation

> US National Institutes of Health, RePORTER application 10833623, The critical roles of (p)ppGpp in homeostasis and antibiotic tolerance in Gram positive bacteria (5R35GM127088-07). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10833623. Licensed CC0.

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