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.

Highlights d Diverse stress conditions induce transcription of bacterial toxin-antitoxin systems d Growth and RNA-seq assays demonstrate that stress does not activate toxins d Stress can trigger antitoxin degradation and relief from transcriptional repression d New antitoxin synthesis and proteolytically stable TA complexes prevent toxin release

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.

Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.