The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species.

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defense genes in bacterial genomes. This search identified homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADPribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

SummaryThe ancient, ongoing coevolutionary battle between bacteria and their viral predators, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Dozens of additional anti-phage systems have been identified based on their co-location within so-called defense islands, but whether these computational screens are exhaustive is unclear. Here, we developed an experimental selection scheme agnostic to genomic context to identify defense systems encoded in 71 diverse E. coli strains. Our results unveil 21 new and conserved defense systems, none of which were previously detected as enriched in defense islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defense systems in E. coli, with defense systems typically carried in specific locations, or hotspots. These hotspots in homologous prophages and mobile genetic elements encode dozens of additional, as-yet uncharacterized defense system candidates. Collectively, our findings reveal an extended landscape of antiviral immunity in E. coli and provide a generalizable approach for mapping defense systems in other species.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.