# Activation of the anti-phage defense DarTG by infected E. coli

> **NIH NIH F32** · WASHINGTON UNIVERSITY · 2024 · $76,756

## Abstract

PROJECT SUMMARY/ABSTRACT
Background: Bacteria possess an extensive set of anti-viral immune systems to block the
intracellular replication of lytic bacterial viruses (bacteriophages or phages). Among these anti-
phage defenses, toxin-antitoxin (TA) systems are common, two-component genetic modules
containing a lethal toxin and its cognate neutralizing anti-toxin. TA systems serve as primed
defenses: expressed before infection, the antitoxin counteracts its toxin until it senses infection
and frees its toxin to block intracellular phage replication and kill the host bacterium.
 Recently, we identified DarTG1 as an anti-phage TA system (LeRoux 2022). DarTG1 consists
of (1) an ADP-ribosylase toxin, DarT1, that modifies single-stranded DNA (ssDNA) to block phage
DNA replication & transcription and (2) a glycohydrolase antitoxin, DarG1, that removes ADP-
ribosylation during normal bacterial growth. Despite knowing how DarTG1 defends against phage,
it is unknown how DarTG1 senses phage infection while avoiding lethal spontaneous activation.
Goal/Preliminary Data: As DarT1 is always active when expressed without DarG1, I hypothesize
that DarG1 senses phage infection. Thus, I aim to elucidate how DarG1 senses viral infection and
stops counteracting DarT1. At present, only two TA systems have known activation mechanisms
during phage infection—host transcriptional shutdown and viral capsid expression—and DarG1
senses neither. Instead, my preliminary data suggest that DarG1 senses phage DNA metabolism.
First, we identified diverse phage DNA metabolism proteins that activate DarT1 in the absence of
infection, indicating that DarG1 senses a DNA-related process. Second, DarG1 physically
associates with E. coli’s single-stranded DNA binding protein (SSB) before infection, suggesting
that DarG1 localizes with SSB to ssDNA found at DNA replication forks and DNA lesions. Thus,
I hypothesize that DarG1 monitors ssDNA metabolism for infection-induced perturbations.
Approach: I will determine which phage DNA metabolism processes co-localize with DarG1
during infection and which are necessary for DarG1 to sense infection (aim 1). In parallel, I will
identify which DNA metabolism processes are sufficient for DarG1 to react in uninfected E. coli
by introducing targeted DNA perturbations & known E. coli DNA metabolism proteins (aim 2).
Significance: Uncovering how DarTG1 senses and initiates a response to phage infection will
enhance our understanding of anti-viral immunity, as DarG1-like NADAR-containing proteins are
found across prokaryotes & eukaryotes, suggesting any DarG1 surveillance mechanism identified
may be echoed in other prokaryotic & eukaryotic anti-viral systems.

## Key facts

- **NIH application ID:** 10997202
- **Project number:** 1F32AI186521-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Kyle Dane Gibbs
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $76,756
- **Award type:** 1
- **Project period:** 2024-08-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10997202

## Citation

> US National Institutes of Health, RePORTER application 10997202, Activation of the anti-phage defense DarTG by infected E. coli (1F32AI186521-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10997202. Licensed CC0.

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