Fig 1.
A. Plaque morphology was inconsistent with some bullseye plaques containing 1 mm center clearings with varying degrees of turbidity. Plaque sizes ranged from 2–5 mm in diameter, with an average plaque diameter of approximately 3.5 mm. B. TEM image of BlueFeather at 67,000X magnification. The capsid was estimated to be 48 ± 8 nm and the tail 156 ± 53 nm.
Fig 2.
BlueFeather genome shares little nucleotide similarity but many phams with Cluster FE and former Cluster FI.
The BlueFeather genome is linear with a relatively small length of 16 kbp. Of the 25 identified ORFs, 18 were of known function, 6 were orphams and 1 was a reverse gene. BlueFeather had little BLASTn homology to its most similar phages, as indicated by the limited orange and yellow shading.
Fig 3.
Dot plots suggest shared amino acids but not nucleotides.
Whole genomes and proteomes for each phage were concatenated and dot plots were created using Gepard. Original cluster information is denoted along the top of each figure, with phage BlueFeather indicated by *. A. A whole genome dot plot with word size of 15 indicates strong intracluster nucleotide similarities with both FE and former FI phages. No intercluster nucleotide similarities were observed, indicating BlueFeather does not share significant nucleotide sequences with any of these phages. B. A whole proteome dot plot with a word size of 5 indicated the same intracluster amino acid similarities seen in the genome dot plot, but there were also amino acid similarities observed between BlueFeather, Cluster FE, and former Cluster FI phages. BlueFeather appeared to have greater amino acid similarity with phages originally assigned to Cluster FI.
Fig 4.
BlueFeather shares the most phams with phages originally assigned to Cluster FE and former Cluster FI.
A. Gene Content Similarity (GCS) between BlueFeather, Cluster FE and the former Cluster FI was calculated with the PhagesDB GCS calculator using the number of shared phams. There was high intracluster GCS, and BlueFeather showed higher GCS values with former Cluster FI. B. Gene Content Dissimilarity (GCD) output values of all pairwise comparisons of BlueFeather and all Arthrobacter phages (305), ordered by magnitude. Cluster FE and former Cluster FI were found to be least dissimilar to BlueFeather, with a MaxGCDGap of 41.60%, between BlueFeather and Yavru. (C) GCD output values of all pairwise comparisons of BlueFeather and all phages in PhagesDB (3381). MaxGCDGap remained at 41.60%. There are no non-Arthrobacter phages that are less dissimilar to BlueFeather than Yavru. BlueFeather shares up to 10% of genes with at least 63 non-Arthrobacter phages.
Fig 5.
Expanded Cluster FE includes BlueFeather and former FI phages.
A SplitsTree was generated in order to group Arthrobacter phages based on pham similarity. Ten representative phages from each cluster were selected to measure evolutionary relatedness. While there is great diversity of Arthrobacter phages, BlueFeather forms a relatively small branch with phages originally assigned to Cluster FE and the former Cluster FI. These phages, boxed in blue, comprise the expanded FE Cluster.
Fig 6.
Evidence of horizontal gene transfer in the BlueFeather genome.
The GC content for each gene in BlueFeather’s genome ranged from 59.30%-70.30%, with an average of 64.30%. The number of unique isolation hosts that were represented in each pham ranged from 1–4. Genes with unexpectedly high values were considered to be the result of horizontal gene transfer. There were four genes with the most convincing evidence, indicated by *.