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.

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.

19The alternative sigma factor RpoS is an important regulatory protein in Escherichia coli, 20 responsible for mediating the general stress response. RpoS levels vary continuously in response 21 to different stresses. Previous work has shown that genes vary in their responsiveness to 22 increasing RpoS concentrations, with some genes being "sensitive," requiring only a low level of 23RpoS to be relatively highly expressed, while other genes are "insensitive," only being highly 24 expressed in the presence of high levels of RpoS. In other systems, this type of variation is 25 caused by interactions between the regulatory protein and the DNA it binds. To see if this is the 26 case for RpoS, we measured twelve RpoS binding site mutants for their effects on maximal 27 expression and responsiveness to increasing RpoS concentration. While maximal expression 28 varied over an order of magnitude across these twelve constructs, the responsiveness to 29 increasing RpoS concentration was largely unaffected, suggesting that the RpoS binding site 30 alone is not responsible for a genes' sensitivity or insensitivity to RpoS. In addition, we swapped 31 the RpoS binding region between sensitive and insensitive promoters and found no change in the 32 behavior of the promoter. Taken together, these results argue that differences in sensitivity of 33 the RpoS-dependent promoters are not due to interactions between RpoS and the various DNA 34 sites it binds. 35 36