DNA ADP-ribosylation in the bacterial-phage arms race

ADP-ribosylation is a modification used across domains of life to mediate biological conflicts. The covalent attachment of ADP-ribose to diverse substrates ranging from proteins and small molecules to nucleic acids can render the target inaccessible or inactive. DNA targeting ADP-ribosylation has received less attention than the modification of other substrates, but is increasingly thought to be a widespread strategy in both interbacterial conflicts and in anti-phage defense mechanisms. DNA ADP-ribosylation was discovered in bacteria less than ten years ago, and therefore despite the prevalence of this modification, little is known about its biological function. My postdoctoral research provided a major advance for the field by revealing that a widely distributed bacterial DNA targeting ADP-ribosyltransferase (ART) toxin is the effector of a family of phage defense systems, thus ascribing a clear biological function to these enzymes. DNA targeted ADP-ribosylation blocks DNA replication and is accordingly highly toxic in bacterial cells but also potently anti-viral. These bacterial DNA targeting ARTs, a family termed DarT, are normally kept inactive by a cognate, neutralizing antitoxin, DarG, which is a DNA targeting ADP-ribosylglycohydrolase (ARG). Many fundamental questions remain about the biology of DarTG systems, including how the DarT toxin becomes active after phage infection. Phages, a co-evolving biological entity, are also a rich source of anti-DNA ART mechanisms. We recently discovered that some phages have co-opted DarG-like proteins and related DNA ARGs on multiple occasions, and that these “orphan antitoxins” protect these phages from DarTG-mediated defense. Thus, as with DarT, we were able to ascribe a biological function to a widespread family of previously mysterious phage enzymes. The distribution of DNA ARGs across the tree of life further suggests that DNA ADP-ribosylation is almost certainly more widespread than currently appreciated. The major goals of this study are both to investigate the underlying biology of DarTG systems in their biologically relevant context of phage infection, as well as to develop and apply cutting edge bioinformatic approaches to identify novel DNA ART and ARG families. To this end, we will pursue the following aims: 1) elucidate the molecular mechanism by which phage infection activates DarTG1 using genome-wide, single-cell, and in vitro approaches; 2) investigate the specificity and diversity of phage- encoded DNA ARGs and other phage anti-DNA ART counter-defenses, and 3) mechanistically dissect a third, unstudied DarTG family and develop methods for discovery of additional DarT- and non-DarT-related DNA ARTs. The discoveries we make in this bacterial-phage system, with its powerful experimental and genetic tools, will reveal fundamental facets of DNA ART and ARG biology relevant to the bacterial immune system and lay the groundwork for future studies of anti-viral DNA ADP-ribosylation in eukaryotes. Project Number: 1R01AI195977-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Michele LeRoux | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $638,220 | Activity Code: R01 | Study Section: Prokaryotic Cell and Molecular Biology Study Section[PCMB] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19597701