Novel <i>Erwinia</i> Phage Harboring Glucosylhydroxymethylcytosine DNA Hypermodifications Highlights the First Occurrence Outside <i>Enterobacteriaceae</i>

Background: Erwinia amylovora is a gram-negative phytopathogen and the causative agent of fire blight, which carries a significant economic burden on important food and ornamental crops in the Rosaceae family (e.g., pears and apples). Due to increasingly restrictive regulation of chemical treatments, such as copper sprays and antibiotics, biological control using bacteriophages (phages) has been proposed as a solution. Here, we describe a novel E. amylovora phage Cronus, which has been thoroughly characterized. Materials and Methods: Erwinia phage Cronus was was sequenced and compared to existing phage genomes, the capsids were analyzed using transmission electron microscopy and the host-range was determined using an efficiency of plaquing assay on selected pathogenic E. amylovora isolates. Cronus DNA was also investigated, both by restriction digest and HPLC-MS and shown to encode glucosylhydroxymethylcytosine DNA modifications. The host E. amylovora Ea 1/79 (DSM17948) was also investigated bioinformatically, both for putative anti-phage defense systems and for epigenetic signals, which were linked to predicted methylations using the tools Tombo and NanoDisco. Results: We investigated Cronus for its biocontrol potential, revealing a relatively broad host range on a selection of relevant pathogens of E. amylovora and related species. Importantly and relevant to efficiently overcoming many bacterial defense systems, phage Cronus was shown to have glucosylhydroxymethylcytosine DNA hypermodifications. Finally, the antiphage defense and counterdefense potential of the bacterium and phage were assessed bioinformatically, as well as through the use of third-generation sequencing. This revealed a multitude of putative antiphage defense systems, including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, several restriction-modification systems, phosphorothioate modifications, and more. The presence of these defenses and counterdefenses seems to reflect that of other known Tevenvirinae, with the addition of many novel and less well-described systems. Conclusions: Given the low diversity within E. amylovora in general, the interactions between Cronus and its host could provide important information, which in the future could be used to guide a more targeted and efficient application of phages.