Bacteriophages (phages) exhibit high genetic diversity, and the mosaic nature of the shared genetic pool makes quantifying phage relatedness a shifting target. Early parameters for clustering of related Mycobacteria and Arthrobacter phage genomes relied on nucleotide identity thresholds but, more recently, clustering of Gordonia and Microbacterium phages has been performed according to shared gene content. Singleton phages lack the nucleotide identity and/or shared gene content required for clustering newly sequenced genomes with known phages. Whole genome metrics of novel Arthrobacter phage BlueFeather, originally designated a putative singleton, showed low nucleotide identity but high amino acid and gene content similarity with Arthrobacter phages originally assigned to Clusters FE and FI. Gene content similarity revealed that BlueFeather shared genes with these phages in excess of the parameter for clustering Gordonia and Microbacterium phages. Single gene analyses revealed evidence of horizontal gene transfer between BlueFeather and phages in unique clusters that infect a variety of bacterial hosts. Our findings highlight the advantage of using shared gene content to study seemingly genetically isolated phages and have resulted in the reclustering of BlueFeather, a putative singleton, as well as former Cluster FI phages, into a newly expanded Cluster FE.

Bacteriophages (phages) exhibit high genetic diversity, and the mosaic nature of the shared genetic pool makes quantifying phage relatedness an increasingly difficult task. Singleton phages lack the nucleotide identity and/or shared genes required for clustering with known phages. The goal of this study was to investigate singleton Arthrobacter phage BlueFeather's relationships with similar phages that had been assigned to clusters. Whole genome metrics showed low nucleotide identity, but high amino acid and gene content similarity between BlueFeather and Arthrobacter phage Clusters FE and FI. Gene content similarity revealed that BlueFeather shared genes with Clusters FE and FI in excess of a parameter for clustering Gordonia and Microbacterium phages. Single gene analyses revealed evidence of horizontal gene transfer between BlueFeather and phages in unique clusters that infect a variety of bacterial hosts. Our findings highlight the advantage of using shared gene content to study seemingly genetically isolated phages.