Core Defense Hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems

Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Identifying the full defense repertoire of a given species is still challenging, however. Here, we developed a computational tool to broadly identify anti-phage systems, which was applied to >180,000 genomes available on NCBI, revealing Pseudomonas aeruginosa to possess the most diverse anti-phage arsenal of any species with >200 sequenced genomes. Using network analysis to identify the common neighbors of anti-phage systems, we surprisingly identified two highly conserved core defense hotspot loci (cDHS1 and cDHS2). Across more than 1,000 P. aeruginosa strains, cDHS1 is up to 224 kb (mean: 34 kb) with varied arrangements of at least 31 immune systems while cDHS2 has 24 distinct systems (mean: 15.4 kb). cDHS1/2 are present in most P. aeruginosa isolates, in contrast to highly variable mobile DHSs. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identification of core gene markers that flank immune islands could be a simple approach for immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.