The complex roles of genomic DNA modifications of bacteriophage T4 in resistance to nuclease-based defense systems of E. coli

The interplay between defense and counter-defense systems of bacteria and phages is a major driver of evolution of both organisms, leading to their greatest genetic diversity. Bacterial restriction-modification (R-M) and CRISPR-Cas are two well-known defense systems that target phage DNAs through their nuclease activities, whereas phages have developed counter-defense systems through covalent modifications of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, which leads to the question of whats the role of phage genome modifications in countering these systems. Here, we scanned Escherichia coli genome sequences available in the NCBI databases and found abundant nuclease-containing defense systems, indicating that phage genomic DNA could be a major target for E. coli to restrict infection. From a collection of 816 E. coli strains, we cloned and validated 14 systems. Particularly, Gabija and type III Druantia systems have broad antiphage activities. Using wild-type phage T4 and its mutants, T4 (hmC) and T4 (C), which contain glucosyl-5-hydroxymethylcytosines, 5-hydroxymethylcytosines, and unmodified cytosines in the genomic DNA respectively, we revealed the complex roles of genomic modification of phage T4 in countering the nuclease-containing defense systems other than simply blocking the degradation of genomic DNA by nuclease.