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BlueFeather, the singleton that wasn’t: Shared gene content analysis supports expansion of Arthrobacter phage Cluster FE

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.


Introduction
Bacteriophages are ubiquitous biological entities with an estimated 10 31 phage particles on Earth. Assuming an average length of 200 nm, they would extend 200 million light years if stacked head-to-tail [1]. Phages are found in all ecosystems in which bacteria exist and function as drivers of bacterial evolution [2]. They exhibit horizontal gene transfer (HGT) with each other and with bacteria, resulting in the diverse and mosaic nature of phage genomes [3]. Despite their incredible prevalence in the environment, phages remain largely understudied [4]. Previous research on mycobacteriophages concluded that phages may exhibit a continuum of diversity, wherein genes are constantly being shuffled amongst the phage population, resulting in shared genes and sequences between different clusters [5]. The immense and everexpanding diversity of phage genomes has historically been categorized in terms of nucleotide sequence conservation, with a minimum 50% nucleotide identity and 50% span length to at least one phage in a cluster to warrant membership [6,7]. A mass scale study on Gordonia phages also identified a spectrum of genetic diversity, as clusters did not have clear boundaries [8]. Numerous phages lacked the requirement of 50% nucleotide identity but shared many genes, suggesting a relatedness not captured by nucleotide comparisons alone. This relatedness was confirmed with a gene content network phylogeny, and subsequently the cluster assignment parameter for Gordonia phages [8], and later for Microbacterium phages [9], was adjusted to 35% shared gene content with at least one phage in a cluster. Mycobacteriophages, as well as Gordonia and Microbacterium phages, exhibited this spectrum; however, the extent of diversity varies depending on the current known phage population, which in turn affects how clustering is carried out. Arthrobacter phages were previously found to exchange genes more slowly than Gordonia phages, and the 50% nucleotide clustering parameter was considered sufficient at the time [8]. Further studies on Arthrobacter phages found these phages to be genetically isolated with highly variable gene content for phages that can infect a range of host species. With this great diversity, nucleotide identity was used to separate Arthrobacter phages into 10 distinct clusters and 2 singletons [7], and this parameter has been considered sufficient to categorize the limited number of Arthrobacter phages until recently.
Singleton phages can serve as the seeds to start new clusters or be extremely distinct, as they lack the nucleotide identity and/or shared genes required for clustering with known phages. In this study, the genome of novel Arthrobacter phage BlueFeather was examined for nucleotide and amino acid identity with other known phages. BlueFeather lacked sufficient nucleotide conservation for clustering according to nucleotide-based parameters, and was thus designated a putative singleton. Phage BlueFeather did, however, have notable amino acid conservation and shared gene content with other Arthrobacter phages previously assigned to Clusters FE and FI, suggesting it may not be as isolated as its putative singleton status implied. The outcomes of this research on phage BlueFeather provided evidence for the reclustering of phage BlueFeather, as well as phages formerly assigned to Cluster FI, into a newly expanded Cluster FE.

Sample collection and direct isolation
Soil was collected from Los Angeles, CA in a residential area located at 34.05638889˚N, 118.445010000˚W. Direct isolation of phages was performed by shaking a soil sample and 2X PYCa broth (Yeast Extract 1 g/L, Peptone 15 g/L, 4.5mM CaCl 2 , Dextrose 0.1%) in conical tubes at 250 RPM at 25˚C for 1.5 hours. After incubation, the solution was filtered through a 0.22 μm syringe and spotted onto Arthrobacter globiformis B-2979 (A. globiformis). Plaque purifications were performed as described previously and a high titer lysate was filter-sterilized to be used in subsequent characterization experiments [10]. Representative plaques were measured using ImageJ [11] and average plaque diameter was calculated.

Transmission electron microscopy
Transmission electron microscopy (TEM) was performed on BlueFeather lysate. The sample was placed onto a carbon-coated electron microscope grid and stained with 1% uranyl acetate. Phage particles were visualized using the CM120 Instrument (Philips, Amsterdam, Netherlands), and micrographs were captured. Phage head and tail lengths were measured using ImageJ [11].

Genome sequencing and assembly
Viral DNA was isolated with the Wizard 1 DNA Clean-Up System (cat # A7280, Promega, WI, USA). Sequencing libraries were constructed with the NEBNext 1 Ultra™ II DNA Library Prep kit (New England Biolabs, MA, USA), and sequenced by Illumina-MiSeq at the Pittsburgh Bacteriophage Institute to an approximate shotgun coverage of 3538x. Genome assembly and finishing were performed as previously described [12].

Gene content comparisons
Phage genomes used in this study are available from phagesdb.org [23]. Gepard was used to perform sequence analysis to identify regions of homology between nucleotide sequences or amino acid sequences of different phages [24]. Concatenated whole genome nucleotide and whole proteome amino acid sequences were used to create dot plots with word sizes of 15 and 5, respectively.
SplitsTree was used to generate a network phylogeny in order to reveal the genetic distance between Arthrobacter phages [25]. BlueFeather and up to 10 representative phages from each Arthrobacter cluster were selected from the Actino_Draft database (version 366) for comparison.
The gene content calculator on PhagesDB (https://phagesdb.org/genecontent/) was used to calculate Gene Content Similarity (GCS), the percentage of shared genes in phams (groups of genes with related sequences), between BlueFeather, Cluster FE, and former Cluster FI phages [16]. Gene Content Dissimilarity (GCD) and maximum GCD gap (MaxGCDGap) were calculated using scripts described previously [8]. Heatmaps and scatter plots were created using Prism 8.0.0 (GraphPad Software, San Diego, California, USA) and were used for quantitative analysis and visualization of GCS and GCD values.
PhagesDB Pham View was used to gather information about phages with genes in the same phams as BlueFeather's [23]. PECAAN was used to obtain the nucleotide sequences for each BlueFeather gene (https://discover.kbrinsgd.org). The BiologicsCorp online GC content calculator was used for each gene in the genome (https://www.biologicscorp.com/tools/ GCContent/).

BlueFeather is a siphovirus with a short genome
Phage BlueFeather was isolated from a soil sample via direct isolation on A. globiformis B-2979 at 25˚C and had a mixed plaque size, ranging from 2-5 mm in diameter (average plaque size of approximately 3.5 mm). Plaque morphology was also inconsistent, with some bullseye plaques containing 1 mm center clearings with varying degrees of turbidity ( Fig 1A). Transmission electron microscopy (TEM) at 67,000X magnification showed an average phage capsid diameter and tail length of 48 ± 8 nm and 156 ± 53 nm, respectively ( Fig 1B). The long, flexible, non-contractile tail suggested BlueFeather's classification as a Siphoviridae [26].
BlueFeather's genome had a length of 16,302 bp, 64.30% GC content, and genome ends with 15 base 3' sticky overhangs (CCACGGTTCCCGTCC). Phages that infect Arthrobacter hosts have genome lengths that range from 15,319 bp (Toulouse) to 70,265 bp (PrincessTrina) [7]. The average Arthobacter phage genome length (as of May 2020) was 46,968 bp with a standard deviation of 20,619 bp and a median length of 53,859 bp, suggesting that most Arthrobacter phages have genomes notably larger than that of BlueFeather. BlueFeather's genome contained 25 manually annotated genes; 18 were of known function, 6 were orphams-meaning they have not been identified in any other known phage-and 1 was a reverse gene (Fig 2). The left arm of the genome had highly conserved genes amongst siphoviral Arthrobacter phages, such as those encoding terminase, portal protein, head-to-tail adapter, and tail proteins [7]. Tail tube and sheath genes were absent, confirming the classification of BlueFeather as a siphovirus. Genes characteristic of the lytic life cycle, such as lysin A and holin, were identified; however, there were no genes that would indicate BlueFeather's ability to undergo a lysogenic life cycle, suggesting that BlueFeather is not a temperate phage [27].

Dot plot comparisons revealed synonymous substitutions in BlueFeather's genome
Phage BlueFeather was originally classified as a singleton on PhagesDB due to low nucleotide identity with other known phages. Nucleotide and amino acid dot plots were created to qualitatively compare BlueFeather to the most similar Arthrobacter phages, including those in Cluster FE (Corgi, Idaho, Noely) and former Cluster FI (Whytu, Yavru), as identified by BLASTn. Due to the limited number of sequenced Arthrobacter phages, many of the clusters have few members. Of the 28 Arthrobacter clusters on PhagesDB (as of May 2020), 17 clusters have between 2-4 phages (including the former Cluster FI). As expected, phages originally assigned to the same cluster had alignments indicating large regions of nucleotide similarity [28], while comparison of BlueFeather's genome to phages originally assigned to Clusters FE and FI revealed no homologous sequences (Fig 3A). Unexpectedly, dot plot analysis of concatenated amino acid sequences with a word size of 5 revealed numerous regions of amino acid sequence similarity between these phages (Fig 3B). This reflects, at present, perhaps one of the clearest examples in which a group of phages lack nucleotide identity while sharing considerable amino acid identity.

Gene similarity demonstrates a close relationship between BlueFeather and phages of Cluster FE and former Cluster FI
Gene Content Similarity (GCS) is a key metric in quantifying phage genetic relationships and is calculated by averaging the number of shared genes between two phages [29]. GCS was calculated for BlueFeather, Cluster FE phages, and phages originally assigned to Cluster FI. Blue-Feather shared over 35% of genes with all Cluster FE phages, and over 55% of genes with the former Cluster FI phages. Over 35% of genes were shared in each pairwise comparison performed ( Fig 4A). Given that BlueFeather was originally determined to be a singleton, it was surprising to find GCS greater than the recently adopted threshold of 35% for clustering other phage populations [8,9]. Gene Content Dissimilarity (GCD) is the opposite of GCS and was used to calculate the maximum GCD gap (MaxGCDGap), a metric that represents the degree of isolation between a phage and a selected phage population [8]. GCD was calculated for Blue-Feather and all Arthrobacter phages. There was a MaxGCDGap of 41.60% between BlueFeather  and Yavru, indicating a relatively high degree of separation between BlueFeather and the rest of the Arthrobacter phage population (Fig 4B). Arthrobacter phages exhibiting pairwise GCD values with BlueFeather of less than 1 were found in Clusters AN, AU, AM, AZ, AV, AL, FE, AO, FH, FF, and former Cluster FI, indicating shared gene content. GCD was then calculated for BlueFeather and all known phages in the PhagesDB Actino_draft database (Fig 4C). Similar to the Arthrobacter GCD plot, phages assigned to Cluster FE and former Cluster FI were the least dissimilar to BlueFeather. It is notable that in this comparison, there were 63 additional phages ranging from 0.959 to 0.975 GCD, meaning BlueFeather shares a low number of genes with many non-Arthrobacter phages. Non-Arthrobacter phages exhibiting pairwise GCD values with BlueFeather of less than 1 were found in Microbacterium phage Cluster EE, Mycobacterium phage Clusters N, I, P, and the singleton IdentityCrisis, as well as Gordonia phage Clusters DT, CW and the singleton GMA4.
To compare the relationships between the Arthrobacter phage population as whole and the phages assigned to Cluster FE, former Cluster FI, and BlueFeather, a SplitsTree network phylogeny of the phams from each Arthobacter phage cluster was generated to examine the genetic distance between the phages. As expected, BlueFeather was shown to be more genetically similar to phages originally assigned to Clusters FE and FI than to any other Arthrobacter phage clusters (Fig 5). BlueFeather demonstrated a closer pham similarity to former Cluster FI phages Whytu and Yavru than to Cluster FE phages Idaho, Noely and Corgi; however, these phages altogether formed a distinct branch from the rest of the phages sampled and together comprise the newly expanded Cluster FE.

BlueFeather genome exhibits evidence of horizontal gene transfer
Given that BlueFeather shares genes with phages infecting distinct hosts, we investigated its genome for potential evidence of horizontal gene transfer (HGT). A whole genome heatmap was created using common metrics for evidence of HGT for each gene in the genome. As of March 2020, 4 genes in BlueFeather were considered to have the most convincing evidence for HGT based on GC content and prevalence in phages that infect unique bacterial hosts: genes 2, 15, 19, and 24 (Fig 6).
Typically, viral genes have about the same [30] or slightly lower GC content [31] compared to their bacterial hosts, suggesting that genes with higher GC content may have been horizontally transferred. BlueFeather had an overall average GC content of 64.30% and Arthrobacter globiformis mrc11 was found to have an overall GC content of 65.9% [32]. BlueFeather genespecific average GC contents ranged from 59.30% to 70.30%, and genes with maximum average GC contents were considered for HGT. This included genes 15 and 24 with GC contents of 70.3% and 70.1%, respectively.
It is increasingly understood that phages infecting different hosts may share considerable gene content through processes such as HGT [8]. For each gene in the BlueFeather genome, we calculated the number of unique isolation hosts for phages possessing a pham found in BlueFeather. Gene 2 belongs to a pham with member genes found in phages that infect Gordonia malaquae BEN700 and Arthrobacter sp. ATCC 21022. Gene 15 belongs to a pham with member genes found in phages that infect A. globiformis B-2979, A. sp. ATCC 21022, Mycobacterium smegmatis mc 2 155, G. malaquae BEN700, and Gordonia rubripertincta NRRL B-16540. Gene 19 was the only reverse gene in the BlueFeather genome, and this gene was only found in BlueFeather and in phages infecting Microbacterium foliorum NRRL B-24224 SEA and Microbacterium paraoxydans NWU1.

Discussion
Our research was focused on the genomic and evolutionary relationships between the novel Arthrobacter phage BlueFeather and other known phages, particularly those originally assigned to Clusters FE and FI. Previous studies have shown that new clusters can be formed  when novel phages are found to be similar to former singletons, as demonstrated by the formation of Cluster AS from Arthrobacter singleton Galaxy [7]. BlueFeather, originally designated as a putative singleton phage, exhibits over 35% GCS with all phages originally in Cluster FE and over 55% GCS with those formerly assigned to Cluster FI. The conservation of amino acids, rather than nucleotides, suggests a history of purifying selection via many synonymous mutations in which deleterious mutations were filtered out [33]. Moreover, this presents some of the clearest evidence to date of highly conserved amino acid sequences despite the absence of significant nucleotide conservation amongst related phages.
There is a high degree of synteny between these phages as well. While BlueFeather's original designation as a singleton would imply low genomic relatedness to other phages [5], gene similarity between BlueFeather and phages from Cluster FE and former Cluster FI-in excess of 35%-indicates conservation of gene functions and genome architecture despite extensive divergence of nucleotide identity. While Arthrobacter phages have been clustered according to nucleotide identity in the past [7], this study on BlueFeather, Cluster FE, and the former Cluster FI highlights the importance of continually reevaluating clustering parameters, particularly when different parameters may result in different cluster assignments. Moreover, BlueFeather has the smallest genome of all Arthrobacter singletons, and it is possible that clustering parameters may also need to take genome size into account. Given that GCS reflects the number of genes shared as a proportion of the total number of genes for each phage, the same number of shared genes would yield higher GCS in comparisons between smaller genomes.
Gene content dissimilarity demonstrated that BlueFeather has a MaxGCDGap of 41.60% with phage Yavru, which was originally assigned to Cluster FI. BlueFeather was found to be least dissimilar with Cluster FE and former Cluster FI phages; this was supported by a network phylogeny of representative Arthrobacter phages that indicated great diversity between clusters, but revealed that phage BlueFeather forms a distinct branch with Cluster FE and former Cluster FI phages. Additionally, many phages were found to share between 0-10% GCS with BlueFeather. While this is too low to warrant a significant phylogenetic relationship, it reinforced the observed continuum of diversity in phage populations. Previous research found Arthrobacter phage clusters to be very discrete [7]. Even so, this low yet seemingly widespread display of shared genes, as well as BlueFeather's unexpected relationships with Cluster FE and former Cluster FI phages, provides new insight into the genetic landscape of Arthrobacter phages. Few phages were previously assigned to Cluster FE and the former Cluster FI, representing only 5 of the 306 sequenced and manually annotated Arthrobacter phages (as of May 2020). On the other hand, there are 1,906 sequenced Mycobacterium phages (as of May 2020), which has allowed for a more thorough investigation of the mycobacteriophage continuum of diversity. As more Arthrobacter phages are sequenced, we expect to observe similar trends in these host-dependent genetic landscapes.
Unlike singleton phages that are replete with orphams [5,7], the BlueFeather genome, originally designated as a putative singleton, is composed predominantly of genes with known functions that have been assigned to phams. BlueFeather has less than half as many genes as current Arthrobacter singletons and contains highly conserved genes required for viral mechanisms. These vital functional genes have been more thoroughly studied and as a result, are more likely to be found in phams with predicted functions [16]. Additionally, given that pham assignments are performed on the basis of amino acid identity, it is unsurprising that many of the phams containing these vital functional genes are shared amongst BlueFeather, Cluster FE, and former Cluster FI phages, despite the lack of significant nucleotide identity in gene encoding sequences.
Markers of horizontal gene transfer (HGT) included unexpectedly high GC content, as well as multiple bacterial hosts on which phages sharing genes with BlueFeather were isolated [5].
BlueFeather shared phams with a multitude of non-Arthrobacter phages from various clusters, which allowed us to identify multiple regions as having evidence for HGT. These potential HGT events serve to magnify phage diversity and promote the phenomenon of genetic mosaicism. BlueFeather serves as yet another example of the highly intricate mosaic relationships which exist among phages and are a common feature of the genetic landscape, making phage taxonomy an increasingly difficult task.
In sum, this research has led to the reclustering of BlueFeather and phages formerly assigned to Cluster FI into a newly expanded Cluster FE. Recent observations in which there appear to be limited nucleotide conservation but high shared gene content, as observed in this newly expanded cluster, support the notion that clustering methods should be continually reevaluated and optimized as more phages are sequenced [8]. This study thus provides valuable insight into the continuum of diversity amongst Arthrobacter phages, while also supporting a 35% shared gene content clustering parameter as was previously adopted for Gordonia and Microbacterium phages [8,9]. Further investigation into novel phages is essential to understand the complex phage landscape. As more Arthrobacter phages are discovered, it is likely that we will discover many more phages like BlueFeather which belong to clusters whose close relationships become apparent only through the lens of shared gene content.