Six double-stranded DNA Streptomyces bacteriophages, HotFries, Moozy, RavenPuff, Scap1, Rainydai, and SenditCS, were isolated using the phytopathogen Streptomyces scabiei as a host. These phages have been identified as Siphoviridae and members of cluster BI by genomic analysis.

Bacteriophages are known to display a broad range of host spectra, typically infecting a small set of related bacterial species. The transfer of bacteriophages between more distant clades of bacteria has often been postulated, but remains mostly unaddressed. In this work we leverage the sequencing of novel cluster of phages infecting Streptomyces bacteria and the availability of large numbers of complete phage genomes in public repositories to address this question. Using phylogenetic and comparative genomics methods, we show that several clusters of Actinobacteria-infecting phages are more closely related between them, and with a small group of Firmicutes phages, than with any other Actinobacteriophage lineage. These data indicate that this heterogeneous group of phages shares a common ancestor with well-defined genome structure. Analysis of genomic %GC content shows that these Actinobacteriophages are poorly adapted to their Actinobacteria hosts, suggesting that this phage lineage originated in an ancestor of the Firmicutes, adapted to high %GC content members of this phylum and later migrated to the Actinobacteria.

There is widespread interest in using obligately lytic bacteriophages (“phages”) to treat human bacterial infections. Among Staphylococcus aureus infections, the USA300 lineage is a frequent cause of invasive disease. We observed that phage K, a model S. aureus myophage, exhibits temperature-sensitive growth on USA300 strains, with the wild-type phage providing poorer growth suppression in broth and forming smaller and fainter plaques at 37 °C vs. 30 °C. We isolated 65 mutants of phage K that had improved plaquing characteristics at 37 °C when compared to the parental phage. In all 65 mutants, this phenotype was attributable to loss-of-function (LoF) mutations in gp102, which encodes a protein of unknown function that has homologs only among the Herelleviridae (SPO1-like myophages infecting gram-positive bacteria). Additional experiments with representative mutants consistently showed that the temperature-sensitive plaque phenotype was specific to USA300 MRSA strains and that Gp102 disruption was correlated with improved suppression of bacterial growth in broth and improved antibacterial activity in a mouse model of upper respiratory tract infection. The same genotype and in vitro phenotypes could be replicated in close relatives of phage K. Gp102 disruption did not have a detectable effect on adsorption but did delay cell culture lysis relative to wild-type under permissive infection conditions, suggesting that gp102 conservation might be maintained by selective pressure for more rapid replication. Expression of gp102 on a plasmid was toxic to both an MSSA and a USA300 MRSA strain. Molecular modeling predicts a protein with two helix-turn-helix domains that displays some similarity to DNA-binding proteins such as transcription factors. While its function remains unclear, gp102 is a conserved gene that is important to the infection process of Kayvirus phages, and it appears that the manner in which USA300 strains defend against them at 37 °C can be overcome by gp102 LoF mutations.

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