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.