We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/e139f5c53cffc0375844c618dd7b71e9.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24-48 hours at 30°C.
","imageTitle":"Morphologies of four cluster AS3 phage
","methods":"","reagents":"","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contributes toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russel et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3’ single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_databases) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. All software programs used default settings, unless otherwise specified.
The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were on one side of a bi-directional reporter, with exit-related genes (cro-like protein, excise) immediately downstream on the opposite strand. No tRNAs or orphams were present.
A region with 38 bp conserved homology to the 3’ end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp26 and gp27 on phage Atlantica (positions 19,783…19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS and is homologous to the putative attP site for the related A. globiformis B-2979 phage Galaxy (subcluster AS1) (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
Table 1. Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell.
| Atlantica | Babushka | DanHam62 | Glotell |
Soil sample | Sandy, dry soil outside a garden | Moist soil from a backyard | Moist soil from a backyard | Dry soil/clay sample from a backyard |
Latitude | 33.79672 N | 40.73135 N | 40.73652 N | 33.29193 N |
Longitude | 84.32394 W | 73.72391 W | 73.70929 W | 84.7119 W |
Environment Temp (C) | 23 | 25 | 25 | N/A |
Isolation Temp (C) | 30 | 30 | 30 | 30 |
Plaque Diameter (mm, range) | 1-2 (n=8) | 2-6 | 2-6 | 0.5-1.5 (n=30) |
Tail length (nm, mean ± SD) | 134 ± 3.5 (n=4) | 126.4 ± 2.3 (n=3) | 124.31 ± 3.9 (n=3) | 131.76 ± 2.36 (n=3) |
Capsid Diameter | 62 ± 1.5 (n=5) | 56.14 ± 1.8 (n=3) | 50.1 ± 1.8 (n=3) | 61.76 ± 0.70 (n=3) |
DNA Isolation | Based on (Sobolewski et al., 2022) | Promega Wizard DNA Clean-Up kit | Promega Wizard DNA Clean-Up kit | Promega Wizard DNA Clean-Up kit |
Number of Reads | 3.7 million | 1.5 million | 1.25 million | 0.98 million |
Sequencing Coverage | 9,427 | 3,677 | 3,130 | 2,501 |
Genome Size (bp) | 38,468 | 38,467 | 38.380 | 38,515 |
Genome %GC | 66.1 | 66.0 | 66.0 | 66.1 |
Genome Termini | 3’ Sticky Overhang (12 bp, GAGTTGCCGGCA) | 3’ Sticky Overhang (12 bp, GAGTTGCCGGCA) | 3’ Sticky Overhang (12 bp, GAGTTGCCGGCA) | 3’ Sticky Overhang (12 bp, GAGTTGCCGGCA) |
Number of genes | 69 | 66 | 69 | 71 |
Number of genes with putative functions | 43 (63%) | 43 (65%) | 45 (65%) | 39 (54.9%) |
Genome Accession | PX136956 | PV915904 | PV940979 | PV876937 |
SRA | SRX28484005 | SRX28484006 | SRX28484042 | SRX28484005 |
","references":[{"reference":"
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","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence and Characteristics of Cluster AS3 Arthrobacter globiformis Phages Atlantica, Babushka, DanHam62, and Glotell
","reviews":[{"reviewer":{"displayName":"Sara Tolsma"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Marcie Warner"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"66d21203-df82-4dae-a683-c5e80e6332dc","decision":"edit","abstract":"We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/ce1c8395f4b7068099a22641c8ff8f59.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24 hours (Atlantica) or 48 hours (Babushka, DanHam62, Glotell) at 30°C. Imaging was carried out at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University). (I) Alignment of the putative attP site and upstream region on the phage Atlantica genome with the corresponding regions on the genomes of Babushka, DanHam62, and Glotell. The homologous region on the genome of phage Melons (AS2) and the putative attP site of phage Galaxy (AS1; genome coordinates 20,716–20,755) are shown to illustrate conservation. Multi-sequence alignment was carried out using NCBI BLAST with default parameters for somewhat similar sequences (blastn), and the alignment was visualized in Jalview 2.11.5.1.
Table: Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","imageTitle":"Traits of four cluster AS3 phage
","methods":"N/A
","reagents":"N/A
","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contribute toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russell et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3’ single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_v3.19) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. Sequence alignments were created with BLASTN and visualized in Jalview 2.11.5.1 (Waterhouse et al., 2009). All software programs used default settings, unless otherwise specified.
All genomes were approximately 38.5 kb in length (Table 1) with a 3’ sticky overhang at the genome ends (12 bp, GAGTTGCCGGCA). The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a putative bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were among the alternate-strand genes, with exit-related genes (cro-like protein, excise) immediately downstream, reading in the primary direction. No tRNAs were present. One orpham was identified in the genome of Babushka (stop 37,513).
A region with 38 bp conserved homology to the 3’ end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp27 and gp28 on phage Atlantica (coordinates 19,783–19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS (Fig. 1I) and is homologous to the putative attP site for the related A. globiformis phage Galaxy from subcluster AS1 (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
Table 1. Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
| Atlantica | Babushka | DanHam62 | Glotell | |||||||||||||
Soil sample | Sandy, dry soil outside a garden | Moist soil from a backyard | Moist soil from a backyard | Dry soil/clay sample from a backyard | |||||||||||||
Location Found | Atlanta, GA | New York, NY | New York, NY | Atlanta, GA | |||||||||||||
Latitude | 33.79672 N | 40.73135 N | 40.73652 N | 33.29193 N | |||||||||||||
Longitude | 84.32394 W | 73.72391 W | 73.70929 W | 84.7119 W | |||||||||||||
Environment Temp (C) | 23 | 25 | 25 | N/A | |||||||||||||
Plaque Diameter (mm, range) | 1-2 (n=8) | 2-6 (n-10) | 2-6 (n=10) | 0.5-1.5 (n=30) | |||||||||||||
Tail length (nm, mean ± SD) | 134 ± 3.5 (n=4) | 126.4 ± 2.3 (n=3) | 124.31 ± 3.9 (n=3) | 131.76 ± 2.36 (n=3) | |||||||||||||
Capsid Diameter | 62 ± 1.5 (n=5) | 56.14 ± 1.8 (n=3) | 50.1 ± 1.8 (n=3) | 61.76 ± 0.70 (n=3) | |||||||||||||
DNA Isolation | Based on (Sobolewski et al., 2022) | Promega Wizard DNA Clean-Up kit | Promega Wizard DNA Clean-Up kit | Promega Wizard DNA Clean-Up kit | |||||||||||||
Number of Reads | 3.7 million | 1.5 million | 1.25 million | 0.98 million | |||||||||||||
Sequencing Coverage | 9,427 | 3,677 | 3,130 | 2,501 | |||||||||||||
Genome Size (bp) | 38,468 | 38,467 | 38,380 | 38,515 | |||||||||||||
Genome %GC | 66.1 | 66.0 | 66.0 | 66.1 | |||||||||||||
Number of genes | 69 | 66 | 69 | 71 | |||||||||||||
Number of genes with putative functions | 43 (63%) | 43 (65%) | 45 (65%) | 39 (54.9%) | |||||||||||||
Genome Accession | PX136956 | PV915904 | PV940979 | PV876937 | |||||||||||||
SRA | SRX28484005 | SRX28484006 | SRX28484042 | SRX28484005 |
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","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence and Characteristics of Cluster AS3 Arthrobacter globiformis Phages Atlantica, Babushka, DanHam62, and Glotell
","reviews":[{"reviewer":{"displayName":"Marcie Warner"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"38e75771-28bb-4fba-860c-d6fc99af130b","decision":"edit","abstract":"We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/82dfa1dc5620d6b6c811dc94f4853b13.csv"},"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/ce1c8395f4b7068099a22641c8ff8f59.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24 hours (Atlantica) or 48 hours (Babushka, DanHam62, Glotell) at 30°C. Imaging was carried out at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University). (I) Alignment of the putative attP site and upstream region on the phage Atlantica genome with the corresponding regions on the genomes of Babushka, DanHam62, and Glotell. The homologous region on the genome of phage Melons (AS2) and the putative attP site of phage Galaxy (AS1; genome coordinates 20,716–20,755) are shown to illustrate conservation. Multi-sequence alignment was carried out using NCBI BLAST with default parameters for somewhat similar sequences (blastn), and the alignment was visualized in Jalview 2.11.5.1.
Table: Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","imageTitle":"Traits of four cluster AS3 phage
","methods":"N/A
","reagents":"N/A
","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contribute toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russell et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3' single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_v3.19) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. Sequence alignments were created with BLASTN and visualized in Jalview 2.11.5.1 (Waterhouse et al., 2009). All software programs used default settings, unless otherwise specified.
All genomes were approximately 38.5 kb in length (Table 1) with a 3' sticky overhang at the genome ends (12 bp, GAGTTGCCGGCA). The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a putative bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were among the alternate-strand genes, with exit-related genes (cro-like protein, excise) immediately downstream, reading in the primary direction. No tRNAs were present. One orpham was identified in the genome of Babushka (stop 37,513).
A region with 38 bp conserved homology to the 3' end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp27 and gp28 on phage Atlantica (coordinates 19,783–19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS (Fig. 1I) and is homologous to the putative attP site for the related A. globiformis phage Galaxy from subcluster AS1 (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
Table 1. Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Besemer J. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Research 29: 2607-2618.
","pubmedId":"","doi":"10.1093/nar/29.12.2607"},{"reference":"Conn HJ. 1948. THE MOST ABUNDANT GROUPS OF BACTERIA IN SOIL. Bacteriological Reviews 12: 257-273.
","pubmedId":"","doi":"10.1128/br.12.3.257-273.1948"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Echols H, Murialdo H. 1978. Genetic map of bacteriophage lambda. Microbiological Reviews 42: 577-591.
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","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, Dynamics, and Applications. Annual Review of Virology 7: 37-61.
","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. 2017. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal 11: 1511-1520.
","pubmedId":"","doi":"10.1038/ismej.2017.16"},{"reference":"Jackson, V., & Vega, N. (2025). Successful Lysogen Formation for Cluster AS3 Phage, Atlantica [Dataset]. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. https://doi.org/10.25334/MF8G-3507
","pubmedId":"","doi":"10.25334/MF8G-3507"},{"reference":"Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, et al., Hatfull. 2014. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students. mBio 5: 10.1128/mbio.01051-13.
","pubmedId":"","doi":"10.1128/mbio.01051-13"},{"reference":"Klyczek KK, Bonilla JA, Jacobs-Sera D, Adair TL, Afram P, Allen KG, et al., Hatfull. 2017. Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages. PLOS ONE 12: e0180517.
","pubmedId":"","doi":"10.1371/journal.pone.0180517"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell, D. A. (2024). DOGEMS - Deconvolution of Genomes after En Masse Sequencing. The Actinobacteriophage Database | Blog. https://phagesdb.org/blog/posts/29/
","pubmedId":"","doi":""},{"reference":"Sobolewski I, Adamowicz K, Struck A, Zylicz-Stachula A, Skowron PM. 2022. A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus. Microorganisms 10: 1782.
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","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A. 2015. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Research 43: W401-W407.
","pubmedId":"","doi":"10.1093/nar/gkv485"},{"reference":"Tuttle MJ, Buchan A. 2020. Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence. Environmental Microbiology 22: 4919-4933.
","pubmedId":"","doi":"10.1111/1462-2920.15233"},{"reference":"Waterhouse AM, Procter JB, Martin DMA, Clamp Ml, Barton GJ. 2009. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189-1191.
","pubmedId":"","doi":"10.1093/bioinformatics/btp033"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al., Alva. 2018. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. Journal of Molecular Biology 430: 2237-2243.
","pubmedId":"","doi":"10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence and Characteristics of Cluster AS3 Arthrobacter globiformis Phages Atlantica, Babushka, DanHam62, and Glotell
","reviews":[],"curatorReviews":[]},{"id":"ab99b2cf-b09d-4470-8324-9e9f62cfef40","decision":"accept","abstract":"We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/82dfa1dc5620d6b6c811dc94f4853b13.csv"},"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/ce1c8395f4b7068099a22641c8ff8f59.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24 hours (Atlantica) or 48 hours (Babushka, DanHam62, Glotell) at 30°C. Imaging was carried out at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University). (I) Alignment of the putative attP site and upstream region on the phage Atlantica genome with the corresponding regions on the genomes of Babushka, DanHam62, and Glotell. The homologous region on the genome of phage Melons (AS2) and the putative attP site of phage Galaxy (AS1; genome coordinates 20,716–20,755) are shown to illustrate conservation. Multi-sequence alignment was carried out using NCBI BLAST with default parameters for somewhat similar sequences (blastn), and the alignment was visualized in Jalview 2.11.5.1.
Table: Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","imageTitle":"Traits of four cluster AS3 phage
","methods":"N/A
","reagents":"N/A
","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contribute toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russell et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3' single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_v3.19) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. Sequence alignments were created with BLASTN and visualized in Jalview 2.11.5.1 (Waterhouse et al., 2009). All software programs used default settings, unless otherwise specified.
All genomes were approximately 38.5 kb in length (Table 1) with a 3' sticky overhang at the genome ends (12 bp, GAGTTGCCGGCA). The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a putative bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were among the alternate-strand genes, with exit-related genes (cro-like protein, excise) immediately downstream, reading in the primary direction. No tRNAs were present. One orpham was identified in the genome of Babushka (stop 37,513).
A region with 38 bp conserved homology to the 3' end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp27 and gp28 on phage Atlantica (coordinates 19,783–19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS (Fig. 1I) and is homologous to the putative attP site for the related A. globiformis phage Galaxy from subcluster AS1 (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Besemer J. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Research 29: 2607-2618.
","pubmedId":"","doi":"10.1093/nar/29.12.2607"},{"reference":"Conn HJ. 1948. THE MOST ABUNDANT GROUPS OF BACTERIA IN SOIL. Bacteriological Reviews 12: 257-273.
","pubmedId":"","doi":"10.1128/br.12.3.257-273.1948"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Echols H, Murialdo H. 1978. Genetic map of bacteriophage lambda. Microbiological Reviews 42: 577-591.
","pubmedId":"","doi":"10.1128/mr.42.3.577-591.1978"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, Dynamics, and Applications. Annual Review of Virology 7: 37-61.
","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. 2017. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal 11: 1511-1520.
","pubmedId":"","doi":"10.1038/ismej.2017.16"},{"reference":"Jackson, V., & Vega, N. (2025). Successful Lysogen Formation for Cluster AS3 Phage, Atlantica [Dataset]. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. https://doi.org/10.25334/MF8G-3507
","pubmedId":"","doi":"10.25334/MF8G-3507"},{"reference":"Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, et al., Hatfull. 2014. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students. mBio 5: 10.1128/mbio.01051-13.
","pubmedId":"","doi":"10.1128/mbio.01051-13"},{"reference":"Klyczek KK, Bonilla JA, Jacobs-Sera D, Adair TL, Afram P, Allen KG, et al., Hatfull. 2017. Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages. PLOS ONE 12: e0180517.
","pubmedId":"","doi":"10.1371/journal.pone.0180517"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell, D. A. (2024). DOGEMS - Deconvolution of Genomes after En Masse Sequencing. The Actinobacteriophage Database | Blog. https://phagesdb.org/blog/posts/29/
","pubmedId":"","doi":""},{"reference":"Sobolewski I, Adamowicz K, Struck A, Zylicz-Stachula A, Skowron PM. 2022. A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus. Microorganisms 10: 1782.
","pubmedId":"","doi":"10.3390/microorganisms10091782"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A. 2015. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Research 43: W401-W407.
","pubmedId":"","doi":"10.1093/nar/gkv485"},{"reference":"Tuttle MJ, Buchan A. 2020. Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence. Environmental Microbiology 22: 4919-4933.
","pubmedId":"","doi":"10.1111/1462-2920.15233"},{"reference":"Waterhouse AM, Procter JB, Martin DMA, Clamp Ml, Barton GJ. 2009. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189-1191.
","pubmedId":"","doi":"10.1093/bioinformatics/btp033"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al., Alva. 2018. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. Journal of Molecular Biology 430: 2237-2243.
","pubmedId":"","doi":"10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence and Characteristics of Cluster AS3 Arthrobacter globiformis Phages Atlantica, Babushka, DanHam62, and Glotell
","reviews":[],"curatorReviews":[]},{"id":"6eb4cac7-6624-4d6b-a68a-3ba4a9a8b9dc","decision":"edit","abstract":"We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, New York 11568"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman University, Atlanta, GA 30314"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA 30322"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/82dfa1dc5620d6b6c811dc94f4853b13.csv"},"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/ce1c8395f4b7068099a22641c8ff8f59.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24 hours (Atlantica) or 48 hours (Babushka, DanHam62, Glotell) at 30°C. Imaging was carried out at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University). (I) Alignment of the putative attP site and upstream region on the phage Atlantica genome with the corresponding regions on the genomes of Babushka, DanHam62, and Glotell. The homologous region on the genome of phage Melons (AS2) and the putative attP site of phage Galaxy (AS1; genome coordinates 20,716–20,755) are shown to illustrate conservation. Multi-sequence alignment was carried out using NCBI BLAST with default parameters for somewhat similar sequences (blastn), and the alignment was visualized in Jalview 2.11.5.1.
Table: Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","imageTitle":"Traits of four cluster AS3 phage
","methods":"N/A
","reagents":"N/A
","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contribute toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russell et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3' single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_v3.19) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. Sequence alignments were created with BLASTN and visualized in Jalview 2.11.5.1 (Waterhouse et al., 2009). All software programs used default settings, unless otherwise specified.
All genomes were approximately 38.5 kb in length (Table 1) with a 3' sticky overhang at the genome ends (12 bp, GAGTTGCCGGCA). The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a putative bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were among the alternate-strand genes, with exit-related genes (cro-like protein, excise) immediately downstream, reading in the primary direction. No tRNAs were present. One orpham was identified in the genome of Babushka (stop 37,513).
A region with 38 bp conserved homology to the 3' end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp27 and gp28 on phage Atlantica (coordinates 19,783–19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS (Fig. 1I) and is homologous to the putative attP site for the related A. globiformis phage Galaxy from subcluster AS1 (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Besemer J. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Research 29: 2607-2618.
","pubmedId":"","doi":"10.1093/nar/29.12.2607"},{"reference":"Conn HJ. 1948. THE MOST ABUNDANT GROUPS OF BACTERIA IN SOIL. Bacteriological Reviews 12: 257-273.
","pubmedId":"","doi":"10.1128/br.12.3.257-273.1948"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Echols H, Murialdo H. 1978. Genetic map of bacteriophage lambda. Microbiological Reviews 42: 577-591.
","pubmedId":"","doi":"10.1128/mr.42.3.577-591.1978"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, Dynamics, and Applications. Annual Review of Virology 7: 37-61.
","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. 2017. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal 11: 1511-1520.
","pubmedId":"","doi":"10.1038/ismej.2017.16"},{"reference":"Jackson, V., & Vega, N. (2025). Successful Lysogen Formation for Cluster AS3 Phage, Atlantica [Dataset]. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. https://doi.org/10.25334/MF8G-3507
","pubmedId":"","doi":"10.25334/MF8G-3507"},{"reference":"Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, et al., Hatfull. 2014. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students. mBio 5: 10.1128/mbio.01051-13.
","pubmedId":"","doi":"10.1128/mbio.01051-13"},{"reference":"Klyczek KK, Bonilla JA, Jacobs-Sera D, Adair TL, Afram P, Allen KG, et al., Hatfull. 2017. Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages. PLOS ONE 12: e0180517.
","pubmedId":"","doi":"10.1371/journal.pone.0180517"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell, D. A. (2024). DOGEMS - Deconvolution of Genomes after En Masse Sequencing. The Actinobacteriophage Database | Blog. https://phagesdb.org/blog/posts/29/
","pubmedId":"","doi":""},{"reference":"Sobolewski I, Adamowicz K, Struck A, Zylicz-Stachula A, Skowron PM. 2022. A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus. Microorganisms 10: 1782.
","pubmedId":"","doi":"10.3390/microorganisms10091782"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A. 2015. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Research 43: W401-W407.
","pubmedId":"","doi":"10.1093/nar/gkv485"},{"reference":"Tuttle MJ, Buchan A. 2020. Lysogeny in the oceans: Lessons from cultivated model systems and a reanalysis of its prevalence. Environmental Microbiology 22: 4919-4933.
","pubmedId":"","doi":"10.1111/1462-2920.15233"},{"reference":"Waterhouse AM, Procter JB, Martin DMA, Clamp Ml, Barton GJ. 2009. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189-1191.
","pubmedId":"","doi":"10.1093/bioinformatics/btp033"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al., Alva. 2018. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. Journal of Molecular Biology 430: 2237-2243.
","pubmedId":"","doi":"10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence and Characteristics of Cluster AS3 Arthrobacter globiformis Phages Atlantica, Babushka, DanHam62, and Glotell
","reviews":[],"curatorReviews":[]},{"id":"7abb3198-875c-4387-833b-b87fafdbdf8c","decision":"publish","abstract":"We report the discovery and characterization of four phages infecting Arthrobacter globiformis B-2979 that are all assigned to actinobacteriophage subcluster AS3 based on gene content similarity. These phages have genomes of approximately 38 kbp, with 66-71 predicted protein-coding genes and no tRNA genes. Genome architecture suggests that these phage are temperate. An attP core sequence with homology to host tRNA-fMet has been identified.
","acknowledgements":"We thank Daniel Russell and Rebecca Garlena for sequencing and assembling the genome, the SEA MINT team for feedback on the manuscript, Tagide deCarvalho for imaging Glotell, Dr. Ricardo Guerrero-Ferreira and Dr. Ted Whitworth of the Emory University Robert P. Apkarian Integrated Electron Microscopy Core (RRID: SCR_023537) for imaging Atlantica, and Graham Hatfull and the Hatfull lab, the SEA-PHAGES program, and our respective institutions for support. Atlantica was isolated by Valerie Jackson. Babushka was isolated by Leah Joby and Ramanpreet Kaur. DanHam62 was isolated by Hameeda Rasheed and Daniela Jara. Glotell was isolated by Loren Lewis. All authors contributed to the annotation of these genomes.
","authors":[{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"sbakayok@nyit.edu","firstName":"Sarah","lastName":"Bakayoko","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"jchen174@nyit.edu","firstName":"Justin","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"yu-chuan.chen@emory.edu","firstName":"Yu-Chuan","lastName":"Chen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"valerie.jackson@emory.edu","firstName":"Valerie N.","lastName":"Jackson","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"jacob.hillman@emory.edu","firstName":"Jacob","lastName":"Hillman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"djara@nyit.edu","firstName":"Daniela","lastName":"Jara","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ljoby@nyit.edu","firstName":"Leah","lastName":"Joby","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"rkaur73@nyit.edu","firstName":"Ramanpreet","lastName":"Kaur","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"nikhita.lalwani@emory.edu","firstName":"Nikhita","lastName":"Lalwani","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"klarse04@nyit.edu","firstName":"Kirsty","lastName":"Larsen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Spelman College, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"lorenlewis@spelman.edu","firstName":"Loren","lastName":"Lewis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["investigation","writing_originalDraft"],"email":"martin.lin@emory.edu","firstName":"Martin","lastName":"Lin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"hhameeda@nyit.edu","firstName":"Hameeda","lastName":"Rasheed","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"ftariq01@nyit.edu","firstName":"Faizan","lastName":"Tariq","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"cwallis@nyit.edu","firstName":"Chennai","lastName":"Wallis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["New York Institute of Technology, Old Westbury, NY, United States"],"departments":["Department of Biological and Chemical Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"bgibb@nyit.edu","firstName":"Bryan","lastName":"Gibb","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-2094-344X"},{"affiliations":["Spelman College, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"jmelton@spelman.edu","firstName":"James T.","lastName":"Melton III","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3335-5129"},{"affiliations":["Emory University, Atlanta, GA, United States"],"departments":["Department of Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"nic.vega@emory.edu","firstName":"Nic M.","lastName":"Vega","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9929-6109"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/82dfa1dc5620d6b6c811dc94f4853b13.csv"},"extendedData":[],"funding":"Funding for undergraduate lab activities was provided by the authors' respective institutions. Vega was supported in part by NSF award 2340578.
","image":{"url":"https://portal.micropublication.org/uploads/ce1c8395f4b7068099a22641c8ff8f59.png"},"imageCaption":"Plaque morphology (A-D) and negative-staining TEM (E-H) with 1% uranyl acetate of phage Atlantica (A, E), Babushka (B, F), DanHam62 (C, G), and Glotell (D, H). Plaque formation on isolation host B-2979 with 0.2% (Atlantica) or 0.4% PYCa top agar after 24 hours (Atlantica) or 48 hours (Babushka, DanHam62, Glotell) at 30°C. Imaging was carried out at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University). (I) Alignment of the putative attP site and upstream region on the phage Atlantica genome with the corresponding regions on the genomes of Babushka, DanHam62, and Glotell. The homologous region on the genome of phage Melons (AS2) and the putative attP site of phage Galaxy (AS1; genome coordinates 20,716–20,755) are shown to illustrate conservation. Multi-sequence alignment was carried out using NCBI BLAST with default parameters for somewhat similar sequences (blastn), and the alignment was visualized in Jalview 2.11.5.1.
Table: Isolation and characterization of AS3 phage Atlantica, Babushka, DanHam62, and Glotell. Protocols were carried out using the equipment and resources at each originating institution (Atlantica, Emory University; Babushka and DanHam62, NYIT; Glotell, Spelman University).
","imageTitle":"Traits of four cluster AS3 phage
","methods":"N/A
","reagents":"N/A
","patternDescription":"Temperate bacteriophage, which can propagate through vertical transmission with the host, are common, with prophages being detectable in a substantial fraction of bacterial genomes (Tuttle & Buchan, 2020). Temperate phage can substantially affect the ecology and evolution of bacterial populations (Howard-Varona et al., 2017). Here we present the isolation and genome characterization of four genetically similar bacteriophages infecting the common soil actinobacterium Arthrobacter globiformis (Conn, 1948). All four are predicted to be temperate, and one has been demonstrated to establish lysogeny. The data collectively contribute toward a better understanding of phage diversity in this clade of hosts (Hatfull, 2020) and of temperate phage diversity more broadly.
The phages Atlantica, Babushka, DanHam62, and Glotell were extracted from soil samples collected in New York and Georgia, USA (Table 1) as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Jordan et al., 2014) and using common procedures (Zorawik et al., 2024). Briefly, soil samples were washed with peptone-yeast extract-calcium (PYCa) liquid medium and sterile-filtered (0.22 µM pore size). Filtrate was inoculated with A. globiformis B-2979 and incubated with shaking at 200 RPM and 30°C for 48h before centrifugation to pellet bacteria and sterile filtration of supernatant. To create double-layer plates, soft (0.2-0.4%) top agar containing A. globiformis B-2979 and aliquots of filtrates was overlaid on PYCa base agar and incubated for 24-48h at 30°C before viewing plaques. The morphology and diameter of plaques are presented in Table 1 and Figure 1, respectively. Replication of phages produced small to medium-sized, clear to hazy plaques (Fig. 1, Table 1). Phage were purified by 2-3 rounds of picking a single, well-isolated plaque, followed by serial dilution and plating to determine homogeneity. Negative-staining transmission electron microscopy of all phages indicated a short-tailed siphovirus morphology with a nearly isocahedral capsid, consistent with other Caudoviricetes bacteriophages such as phage λ (Fig. 1). Measurements for capsid diameter and tail length can be found in Table 1.
Genomic DNA was purified from high-titer lysates of the purified phages using methods shown in Table 1. Illumina libraries were prepared with the NEB Ultra II FS kit and sequenced on an Illumina NextSeq 1000 (XLEP-P1 kit, single-end 100-bp reads) (Russell, 2018). Glotell was sequenced as part of a DOGEMS (Deconvolution of Genomes after En Masse Sequencing) sample (Russell, 2024). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly. Trimmed reads were assembled per Russell et al. (Russell, 2018), using Newbler v2.9 (Miller et al., 2010) with default parameters, generating single contigs that were verified for completeness and genomic termini using Consed v2.9 (Gordon et al., 1998). The resulting genomes (Table 1) were ~38 kbp. All phages had termini characterized by 12-bp 3' single-stranded overhangs, and GC content was ~66%, similar to that of the host. Phages were assigned to subcluster AS3 based on gene content similarity of at least 35% to phages in the Actinobacteriophage Database (PhagesDB.org) (Pope et al., 2017; Russell & Hatfull, 2017).
The genomes were automatically annotated using DNA Master v5.23.6, build 270 (http://cobamide2.bio.pitt.edu/computer.htm) (Pope & Jacobs-Sera, 2018) and PECAAN v20241104 (discover.kbrinsgd.org) (Rinehart et al., 2016). The initial auto-annotation was performed with Glimmer v3.02b (Delcher et al., 2007) and GeneMark v2 or v4.28 (Besemer et al., 2001; Besemer & Borodovsky, 2005), along with Starterator (http://phages.wustl.edu/starterator/) to help refine predicted gene coordinates. BLAST (Altschul et al., 1990) using the NCBI nonredundant and actinobacteriophage databases, HHPred v2.0.13 (Söding et al., 2005) using PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains (CD_v3.19) (Zimmermann et al., 2018), and Phamerator [Actino_Draft v595 database] (Cresawn et al., 2011) were used as comparative tools to identify the putative gene functions. No tRNAs were identified using Aragorn v1.2.41 (Laslett & Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe & Eddy, 1997). To assess the presence of transmembrane proteins, TMHMM v2.0 (Krogh et al., 2001) and TOPCONS 2.0 (Tsirigos et al., 2015) programs were used. Sequence alignments were created with BLASTN and visualized in Jalview 2.11.5.1 (Waterhouse et al., 2009). All software programs used default settings, unless otherwise specified.
All genomes were approximately 38.5 kb in length (Table 1) with a 3' sticky overhang at the genome ends (12 bp, GAGTTGCCGGCA). The resulting annotations indicated a λ-like genome architecture, with lysogeny-related genes and early genes transcribed on opposite sides of a putative bi-directional promoter (Echols & Murialdo, 1978). These genomes contained 66-71 predicted protein-coding genes, mostly transcribed in a single direction (~80% of genes) with the exception of a short block of genes near the middle of the genome (gp24 to gp36-39). Approximately 60% of genes could be assigned putative functions, including structural genes, lysis genes (endolysin and holin), and genes involved in DNA replication (RecE-like, RecT-like, RepA-like, helicase loader, SSB). Genes involved in lysogeny (Int-Y, immunity regulator) were among the alternate-strand genes, with exit-related genes (cro-like protein, excise) immediately downstream, reading in the primary direction. No tRNAs were present. One orpham was identified in the genome of Babushka (stop 37,513).
A region with 38 bp conserved homology to the 3' end of one of two tandem copies of Arthrobacter tRNA-fMet was identified between hypothetical proteins gp27 and gp28 on phage Atlantica (coordinates 19,783–19,821), several ORFs upstream of Int-Y (Atlantica gp36). BLASTN confirmed that this region is highly conserved within cluster AS (Fig. 1I) and is homologous to the putative attP site for the related A. globiformis phage Galaxy from subcluster AS1 (Klyczek et al., 2017). Integration of Atlantica at the proposed site was confirmed by PCR (Jackson & Vega, 2025).
Nucleotide sequence accession numbers
Complete genome sequences and sequence read archives (SRA) are available at GenBank. Accession numbers and SRA numbers are given in Table 1.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Besemer J. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Research 29: 2607-2618.
","pubmedId":"","doi":"10.1093/nar/29.12.2607"},{"reference":"Conn HJ. 1948. THE MOST ABUNDANT GROUPS OF BACTERIA IN SOIL. Bacteriological Reviews 12: 257-273.
","pubmedId":"","doi":"10.1128/br.12.3.257-273.1948"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Echols H, Murialdo H. 1978. Genetic map of bacteriophage lambda. Microbiological Reviews 42: 577-591.
","pubmedId":"","doi":"10.1128/mr.42.3.577-591.1978"},{"reference":"Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.
","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, Dynamics, and Applications. Annual Review of Virology 7: 37-61.
","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Howard-Varona C, Hargreaves KR, Abedon ST, Sullivan MB. 2017. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal 11: 1511-1520.
","pubmedId":"","doi":"10.1038/ismej.2017.16"},{"reference":"Jackson, V., & Vega, N. (2025). Successful Lysogen Formation for Cluster AS3 Phage, Atlantica [Dataset]. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. https://doi.org/10.25334/MF8G-3507
","pubmedId":"","doi":"10.25334/MF8G-3507"},{"reference":"Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, et al., Hatfull. 2014. A Broadly Implementable Research Course in Phage Discovery and Genomics for First-Year Undergraduate Students. mBio 5: 10.1128/mbio.01051-13.
","pubmedId":"","doi":"10.1128/mbio.01051-13"},{"reference":"Klyczek KK, Bonilla JA, Jacobs-Sera D, Adair TL, Afram P, Allen KG, et al., Hatfull. 2017. Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages. PLOS ONE 12: e0180517.
","pubmedId":"","doi":"10.1371/journal.pone.0180517"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell, D. A. (2024). DOGEMS - Deconvolution of Genomes after En Masse Sequencing. The Actinobacteriophage Database | Blog. https://phagesdb.org/blog/posts/29/
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","pubmedId":"","doi":"10.3390/microorganisms10091782"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
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