Novel bacteriophages Andrew and RedFox were isolated from soil samples collected in New York and Pennsylvania and propagated using Arthrobacter globiformis B-2979. Both phages are assigned to actinobacteriophage subcluster AS3 based on gene content. Although these phages exhibit conserved genomic content characteristics of the AS3 subcluster, there are notable differences. In particular, phage Andrew gene 25 in the central region of the genome and genes 64, 65, and 66 near the end of the genome lack homologs in cluster AS3 or in the actinobacteriophage database more broadly.
","acknowledgements":"We would like to acknowledge John H. Martinson Honors College, Rowan University College of Science and Mathematics, and Dr. Stephen Bentivenga for their continued support.
We would like to acknowledge the University of Pittsburgh and Marist University SEA-PHAGES faculty and students for the discovery of the Andrew and RedFox bacteriophages and the annotation of the bacteriophages’ genomes.
We would also like to acknowledge Dr. Vic Sivinathan and Dr. Graham Hatfull from HHMI’s SEA-PHAGES program.
","authors":[{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"baldwi87@rowan.edu","firstName":"Paige","lastName":"Baldwin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University"],"departments":["Biology"],"credit":["investigation"],"email":"Megan.Dennis@marist.edu","firstName":"Megan","lastName":"Dennis ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikni78@rowan.edu","firstName":"Niran","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikha49@rowan.edu","firstName":"Hanan","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"djs@pitt.edu","firstName":"Deborah","lastName":"Jacobs-Sera ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University"],"departments":["Biology"],"credit":["investigation"],"email":"Juliana.Magna1@marist.edu","firstName":"Juliana","lastName":"Magna ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mazaka27@rowan.edu","firstName":"Mia","lastName":"Mazakas ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University"],"departments":["Biology"],"credit":["investigation"],"email":"Gabrielle.Rice1@marist.edu","firstName":"Gabriella","lastName":"Rice","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"sswerdlow@pitt.edu","firstName":"Sarah","lastName":"Swerdlow ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"mcu7@pitt.edu","firstName":"Megan","lastName":"Ulbrich ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization"],"email":"farberm@rowan.edu","firstName":"Matthew ","lastName":"Farber","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University "],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization","validation","investigation","writing_reviewEditing"],"email":"bogush@rowan.edu","firstName":"Marina","lastName":"Bogush ","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/77f57b1ba96fe6c697de33cc63777e9a.csv"},"extendedData":[],"funding":"This project has been supported by the Department of Biological and Biomedical Sciences, College of Science and Mathematics, Rowan University, Glassboro, New Jersey
","image":{"url":null},"imageCaption":"Sequencing data and genome characteristics
","imageTitle":"Bacteriophages Andrew and RedFox
","methods":"","reagents":"","patternDescription":"Bacteriophages provide a means to modulate bacterial populations and therefore have practical applications in medicine and industry, including the treatment of bacterial infections (Hibstu et al. 2022; Hatfull et al. 2022) and the reservation of food (Imran et al. 2023). Given their abundance and diversity, the isolation and characterization of novel bacteriophages promise to advance their application. Here, we described the isolation and characterization of two bacteriophages using Arthrobacter globiformis, a soil bacterium that is both widely distributed and ecologically significant (Klyczek et al., 2018).
Bacteriophages Andrew and RedFox were isolated by enrichment (Zorawik et al. 2024) from soil samples collected in mildly wet and nutrient-rich environments in Pittsburgh, Pennsylvania (GPS coordinate: 40.445472 N, 79.998708 W) and Poughkeepsie, New York (GPS coordinate: 41.7203 N, 73.9356 W), respectively. Andrew and RedFox soil samples were suspended in peptone-yeast extract-calcium (PYCa) medium and shaken at 250 rpm for 1 and 1.5 hours, respectively. The suspensions were centrifuged, filtered through a 0.22-μm filter, and the filtrate inoculated with Arthrobacter globiformis B-2979 for 48 hours at 30°C. The enriched cultures were then filtered again, and 10 μL of each filtrate was plated in PYCa top agar with A. globiformis and incubated at 30°C for 48 hours. Phages were purified through three successive rounds of plating for isolated plaques. Both Andrew and RedFox produced clear plaques.
Transmission electron microscopy with 1% uranyl acetate staining for Andrew or UranyLess staining for RedFox revealed siphovirus morphologies characterized by icosahedral capsids and flexible tails. DNA was extracted from bacteriophage lysates using the Promega DNA Wizard Cleanup Kit. Sequencing libraries were prepared using the NEB Ultra II FS DNA library kit and run on the Illumina MiSeq platform (v3 reagents), yielding 150-base single-end reads that were assembled into genomes using Newbler and Consed software (Russell 2018). Sequencing data and genome characteristics are presented in Table 1.
Bacteriophage genome annotations were completed using DNA Master v5.23.6 (https://phagesdb.org/DNAMaster/) embedded with Glimmer v3.02 (Delcher et al. 2007) and GeneMark v2.5p (Besemer and Borodovsky 2005). Gene start sites were refined using Starterator v1.2 (http://phages.wustl.edu/starterator/). To identify putative functions of protein-coding genes, several comparative tools were used, all with default parameters for all software. These included Phamerator (Cresawn et al. 2011) with the Actino_draft database v578; BLAST, using the Actinobacteriophage and NCBI non-redundant databases (Altschul et al. 1990); and HHPRED, using the PDB_mmCIF70, Pfam v.36, and NCBI Conserved Domains databases (Söding et al. 2005). Aragorn v1.2.41 (Laslett and Canback 2004) or tRNAscanSE v.2.0 (Lowe and Chan 2016) were used to identify tRNAs.
Andrew has a genome of 38,802 bp, with a GC content of 65.5%. RedFox is 38728 bp long, with a 66.1% GC content. Both have 3′ 12-base single-stranded sticky ends. The GC content is typical of other Arthrobacter bacteriophages, which range from 45.1% to 68.5% (Klyczek et al. 2018; Russell and Hatfull 2017). These two bacteriophages were sorted into cluster AS and subcluster AS3 based on gene content similarity (GCS) of at least 35% to bacteriophages in the Actinobacteriophage database (Russell and Hatfull 2017; Pope et al. 2017).
Genome annotation revealed that Andrew contains 72 predicted genes, 36 of which were assigned putative functions, whereas RedFox contains 71 predicted genes, 40 of which were assigned putative functions (Table1). Neither genome was found to contain tRNAs genes (Table 1). Both Andrew and RedFox demonstrated high GCS of 83.1% and shared highly conserved genome architectures characteristic of the AS3 subcluster (Glaser et al. 2026). Both genomes displayed a modular structure containing conserved regions associated with DNA packaging, virion structure and assembly, lysogeny, and DNA replication (Glaser et al. 2026). Despite this overall conservation, several regions display greater genomic divergence when compared with AS3 cluster phages, including gene 25 in the central region of the Andrew genome and genes 64, 65 and 66 near the end of Andrew genome. These genes lack homologs in the actinobacteriophage database, to date, adding to the diversity of gene sequences uncovered for actinobacteriophages through the isolation and characterization of novel phages.
GenBank accession numbers for Andrew and RedFox are MH834595 and OR195049). SRA accession numbers for Andrew and RedFox are SRX31241826 and SRX20165779.
","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":"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":"Rivka Glaser; Porter, M.; Gibb, B.; Vega, N.; Swerdlow, S.; Klyczek, K.; Denise L Monti (2026). Subcluster AS3 Annotation Report. HHMI Science Education Alliance (SEA) Faculty Group, (Version 2.0). QUBES Educational Resources. doi:10.25334/QB6X-VH05
","pubmedId":"","doi":""},{"reference":"Hatfull GF, Dedrick RM, Schooley RT. 2022. Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine 73: 197-211.
","pubmedId":"","doi":"10.1146/annurev-med-080219-122208"},{"reference":"Hibstu Z, Belew H, Akelew Y, Mengist HM. 2022. Phage Therapy: A Different Approach to Fight Bacterial Infections. Biologics: Targets and Therapy Volume 16: 173-186.
","pubmedId":"","doi":"10.2147/BTT.S381237"},{"reference":"Imran A, Shehzadi U, Islam F, Afzaal M, Ali R, Ali YA, et al., Rasool. 2023. Bacteriophages and food safety: An updated overview. Food Science & Nutrition 11: 3621-3630.
","pubmedId":"","doi":"10.1002/fsn3.3360"},{"reference":"Klyczek KK, Jacobs-Sera D, Adair TL, Adams SD, Ball SL, Benjamin RC, et al., Hatfull. 2018. Complete Genome Sequences of 44\n Arthrobacter\n Phages. Genome Announcements 6: 10.1128/genomea.01474-17.
","pubmedId":"","doi":"10.1128/genomea.01474-17"},{"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, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.
","pubmedId":"","doi":"10.1093/nar/gkw413"},{"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. 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 DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"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 Analysis of Two Arthrobacter Bacteriophages, Andrew and RedFox, from Subcluster AS3 Isolated from Soil in New York and Pennsylvania
","reviews":[],"curatorReviews":[]},{"id":"bcb0b87d-48a0-4cb8-b520-25d002ee8c2c","decision":"revise","abstract":"Novel bacteriophages Andrew and RedFox were isolated from soil samples collected in New York and Pennsylvania and propagated using Arthrobacter globiformis B-2979. Both phages are assigned to actinobacteriophage subcluster AS3 based on gene content. Although these phages exhibit conserved genomic content characteristics of the AS3 subcluster, there are notable differences. In particular, phage Andrew gene 25 in the central region of the genome and genes 64, 65, and 66 near the end of the genome lack homologs in cluster AS3 or in the actinobacteriophage database more broadly.
","acknowledgements":"We would like to acknowledge John H. Martinson Honors College, Rowan University College of Science and Mathematics, and Dr. Stephen Bentivenga for their continued support.
We would like to acknowledge the University of Pittsburgh and Marist University SEA-PHAGES faculty and students for the discovery of the Andrew and RedFox bacteriophages and the annotation of the bacteriophages’ genomes.
We would also like to acknowledge Dr. Vic Sivinathan and Dr. Graham Hatfull from HHMI’s SEA-PHAGES program.
","authors":[{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"baldwi87@rowan.edu","firstName":"Paige","lastName":"Baldwin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Megan.Dennis@marist.edu","firstName":"Megan","lastName":"Dennis ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikni78@rowan.edu","firstName":"Niran","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikha49@rowan.edu","firstName":"Hanan","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"djs@pitt.edu","firstName":"Deborah","lastName":"Jacobs-Sera ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Juliana.Magna1@marist.edu","firstName":"Juliana","lastName":"Magna ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mazaka27@rowan.edu","firstName":"Mia","lastName":"Mazakas ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Gabrielle.Rice1@marist.edu","firstName":"Gabriella","lastName":"Rice","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"sswerdlow@pitt.edu","firstName":"Sarah","lastName":"Swerdlow ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"mcu7@pitt.edu","firstName":"Megan","lastName":"Ulbrich ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization"],"email":"farberm@rowan.edu","firstName":"Matthew ","lastName":"Farber","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization","validation","investigation","writing_reviewEditing"],"email":"bogush@rowan.edu","firstName":"Marina","lastName":"Bogush ","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/77f57b1ba96fe6c697de33cc63777e9a.csv"},"extendedData":[],"funding":"This project has been supported by the Department of Biological and Biomedical Sciences, College of Science and Mathematics, Rowan University, Glassboro, New Jersey
","image":{"url":null},"imageCaption":"Sequencing data and genome characteristics
","imageTitle":"Bacteriophages Andrew and RedFox
","methods":"","reagents":"","patternDescription":"Bacteriophages provide a means to modulate bacterial populations and therefore have practical applications in medicine and industry, including the treatment of bacterial infections (Hibstu et al. 2022; Hatfull et al. 2022) and the reservation of food (Imran et al. 2023). Given their abundance and diversity, the isolation and characterization of novel bacteriophages promise to advance their application. Here, we described the isolation and characterization of two bacteriophages using Arthrobacter globiformis, a soil bacterium that is both widely distributed and ecologically significant (Klyczek et al., 2018).
Bacteriophages Andrew and RedFox were isolated by enrichment (Zorawik et al. 2024) from soil samples collected in mildly wet and nutrient-rich environments in Pittsburgh, Pennsylvania (GPS coordinate: 40.445472 N, 79.998708 W) and Poughkeepsie, New York (GPS coordinate: 41.7203 N, 73.9356 W), respectively. Andrew and RedFox soil samples were suspended in peptone-yeast extract-calcium (PYCa) medium and shaken at 250 rpm for 1 and 1.5 hours, respectively. The suspensions were centrifuged, filtered through a 0.22-μm filter, and the filtrate inoculated with Arthrobacter globiformis B-2979 for 48 hours at 30°C. The enriched cultures were then filtered again, and 10 μL of each filtrate was plated in PYCa top agar with A. globiformis and incubated at 30°C for 48 hours. Phages were purified through three successive rounds of plating for isolated plaques. Both Andrew and RedFox produced clear plaques.
Transmission electron microscopy with 1% uranyl acetate staining for Andrew or UranyLess staining for RedFox revealed siphovirus morphologies characterized by icosahedral capsids and flexible tails. DNA was extracted from bacteriophage lysates using the Promega DNA Wizard Cleanup Kit. Sequencing libraries were prepared using the NEB Ultra II FS DNA library kit and run on the Illumina MiSeq platform (v3 reagents), yielding 150-base single-end reads that were assembled into genomes using Newbler and Consed software (Russell 2018). Sequencing data and genome characteristics are presented in Table 1.
Bacteriophage genome annotations were completed using DNA Master v5.23.6 (https://phagesdb.org/DNAMaster/) embedded with Glimmer v3.02 (Delcher et al. 2007) and GeneMark v2.5p (Besemer and Borodovsky 2005). Gene start sites were refined using Starterator v1.2 (http://phages.wustl.edu/starterator/). To identify putative functions of protein-coding genes, several comparative tools were used, all with default parameters for all software. These included Phamerator (Cresawn et al. 2011) with the Actino_draft database v578; BLAST, using the Actinobacteriophage and NCBI non-redundant databases (Altschul et al. 1990); and HHPRED, using the PDB_mmCIF70, Pfam v.36, and NCBI Conserved Domains databases (Söding et al. 2005). Aragorn v1.2.41 (Laslett and Canback 2004) or tRNAscanSE v.2.0 (Lowe and Chan 2016) were used to identify tRNAs.
Andrew has a genome of 38,802 bp, with a GC content of 65.5%. RedFox is 38728 bp long, with a 66.1% GC content. Both have 3′ 12-base single-stranded sticky ends. The GC content is typical of other Arthrobacter bacteriophages, which range from 45.1% to 68.5% (Klyczek et al. 2018; Russell and Hatfull 2017). These two bacteriophages were sorted into cluster AS and subcluster AS3 based on gene content similarity (GCS) of at least 35% to bacteriophages in the Actinobacteriophage database (Russell and Hatfull 2017; Pope et al. 2017).
Genome annotation revealed that Andrew contains 72 predicted genes, 36 of which were assigned putative functions, whereas RedFox contains 71 predicted genes, 40 of which were assigned putative functions (Table1). Neither genome was found to contain tRNAs genes (Table 1). Both Andrew and RedFox demonstrated high GCS of 83.1% and shared highly conserved genome architectures characteristic of the AS3 subcluster (Glaser et al. 2026). Both genomes displayed a modular structure containing conserved regions associated with DNA packaging, virion structure and assembly, lysogeny, and DNA replication (Glaser et al. 2026). Despite this overall conservation, several regions display greater genomic divergence when compared with AS3 cluster phages, including gene 25 in the central region of the Andrew genome and genes 64, 65 and 66 near the end of Andrew genome. These genes lack homologs in the actinobacteriophage database, to date, adding to the diversity of gene sequences uncovered for actinobacteriophages through the isolation and characterization of novel phages.
GenBank accession numbers for Andrew and RedFox are MH834595 and OR195049). SRA accession numbers for Andrew and RedFox are SRX31241826 and SRX20165779.
","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":"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":"Freifelder D. 1983. Molecular biology: a comprehensive introduction to prokaryotes and eukaryotes. Jones and Bartlett. ISBN 10: 086720012X / ISBN 13: 9780867200126
","pubmedId":"","doi":""},{"reference":"Hatfull GF, Dedrick RM, Schooley RT. 2022. Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine 73: 197-211.
","pubmedId":"","doi":"10.1146/annurev-med-080219-122208"},{"reference":"Hibstu Z, Belew H, Akelew Y, Mengist HM. 2022. Phage Therapy: A Different Approach to Fight Bacterial Infections. Biologics: Targets and Therapy Volume 16: 173-186.
","pubmedId":"","doi":"10.2147/BTT.S381237"},{"reference":"Imran A, Shehzadi U, Islam F, Afzaal M, Ali R, Ali YA, et al., Rasool. 2023. Bacteriophages and food safety: An updated overview. Food Science & Nutrition 11: 3621-3630.
","pubmedId":"","doi":"10.1002/fsn3.3360"},{"reference":"Klyczek KK, Jacobs-Sera D, Adair TL, Adams SD, Ball SL, Benjamin RC, et al., Hatfull. 2018. Complete Genome Sequences of 44\n Arthrobacter\n Phages. Genome Announcements 6: 10.1128/genomea.01474-17.
","pubmedId":"","doi":"10.1128/genomea.01474-17"},{"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, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.
","pubmedId":"","doi":"10.1093/nar/gkw413"},{"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":"Rinehart CA, Gaffney BL, Smith JR, Wood JD. 2016. PECAAN: Phage Evidence Collection and Annotation Network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY.
","pubmedId":"","doi":""},{"reference":"Rivka Glaser; Porter, M.; Gibb, B.; Vega, N.; Swerdlow, S.; Klyczek, K.; Denise L Monti (2026). Subcluster AS3 Annotation Report. HHMI Science Education Alliance (SEA) Faculty Group, (Version 2.0). QUBES Educational Resources. doi:10.25334/QB6X-VH05
","pubmedId":"","doi":"10.25334/QB6X-VH05"},{"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 DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"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 Analysis of Two Arthrobacter Bacteriophages, Andrew and RedFox, from Subcluster AS3 Isolated from Soil in New York and Pennsylvania
","reviews":[{"reviewer":{"displayName":"Sara Tolsma"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"b3ee5bc0-19eb-4e51-b2a2-20fa04bf2ee4","decision":"accept","abstract":"Novel bacteriophages Andrew and RedFox were isolated from soil samples collected in New York and Pennsylvania and propagated using Arthrobacter globiformis B-2979. Both phages are assigned to actinobacteriophage subcluster AS3 based on gene content. Although these phages exhibit conserved genomic content characteristics of the AS3 subcluster, there are notable differences. In particular, phage Andrew gene 25 in the central region of the genome and genes 64, 65, and 66 near the end of the genome lack homologs in cluster AS3 or in the actinobacteriophage database more broadly.
","acknowledgements":"We would like to acknowledge John H. Martinson Honors College, Rowan University College of Science and Mathematics, and Dr. Stephen Bentivenga for their continued support.
We would like to acknowledge the University of Pittsburgh and Marist University SEA-PHAGES faculty and students for the discovery of the Andrew and RedFox bacteriophages and the annotation of the bacteriophages’ genomes.
We would also like to acknowledge Dr. Vic Sivinathan and Dr. Graham Hatfull from HHMI’s SEA-PHAGES program.
","authors":[{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"baldwi87@rowan.edu","firstName":"Paige","lastName":"Baldwin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Megan.Dennis@marist.edu","firstName":"Megan","lastName":"Dennis ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikni78@rowan.edu","firstName":"Niran","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikha49@rowan.edu","firstName":"Hanan","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"djs@pitt.edu","firstName":"Deborah","lastName":"Jacobs-Sera ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Juliana.Magna1@marist.edu","firstName":"Juliana","lastName":"Magna ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mazaka27@rowan.edu","firstName":"Mia","lastName":"Mazakas ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist College, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Gabrielle.Rice1@marist.edu","firstName":"Gabriella","lastName":"Rice","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"sswerdlow@pitt.edu","firstName":"Sarah","lastName":"Swerdlow ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"mcu7@pitt.edu","firstName":"Megan","lastName":"Ulbrich ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization"],"email":"farberm@rowan.edu","firstName":"Matthew ","lastName":"Farber","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization","validation","investigation","writing_reviewEditing"],"email":"bogush@rowan.edu","firstName":"Marina","lastName":"Bogush ","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/17d50d2190b1f1631c6528eb797465bb.csv"},"extendedData":[],"funding":"This project has been supported by the Department of Biological and Biomedical Sciences, College of Science and Mathematics, Rowan University, Glassboro, New Jersey
","image":{"url":null},"imageCaption":"Sequencing data and genome characteristics
","imageTitle":"Bacteriophages Andrew and RedFox
","methods":"","reagents":"","patternDescription":"Bacteriophages provide a means to modulate bacterial populations and therefore have practical applications in medicine and industry, including the treatment of bacterial infections (Hibstu et al. 2022; Hatfull et al. 2022) and the preservation of food (Imran et al. 2023). Given their abundance and diversity, the isolation and characterization of novel bacteriophages promise to advance their application. Here, we described the isolation and characterization of two bacteriophages using Arthrobacter globiformis, a soil bacterium that is both widely distributed and ecologically significant (Klyczek et al., 2018).
Bacteriophages Andrew and RedFox were isolated by enrichment (Zorawik et al. 2024) from soil samples collected in mildly wet and nutrient-rich environments in Pittsburgh, Pennsylvania (GPS coordinate: 40.445472 N, 79.998708 W) and Poughkeepsie, New York (GPS coordinate: 41.7203 N, 73.9356 W), respectively. Andrew and RedFox soil samples were suspended in peptone-yeast extract-calcium (PYCa) medium and shaken at 250 rpm for 1 and 1.5 hours, respectively. The suspensions were centrifuged, filtered through a 0.22-μm filter, and the filtrate inoculated with Arthrobacter globiformis B-2979 for 48 hours at 30°C. The enriched cultures were then filtered again, and 10 μL of each filtrate was plated in PYCa top agar with A. globiformis and incubated at 30°C for 48 hours. Phages were purified through three successive rounds of plating for isolated plaques. Both Andrew and RedFox produced clear plaques.
Transmission electron microscopy with 1% uranyl acetate staining for Andrew or UranyLess staining for RedFox revealed siphovirus morphologies characterized by icosahedral capsids and flexible tails. The corresponding plaque and transmission electron microscopy (TEM) images are available through the PhagesDB entries for Andrew and RedFox, respectively (Russell and Hatfull 2017). DNA was extracted from bacteriophage lysates using the Promega DNA Wizard Cleanup Kit. Sequencing libraries were prepared using the NEB Ultra II FS DNA library kit and run on the Illumina MiSeq platform (v3 reagents), yielding 150-base single-end reads that were assembled into genomes using Newbler and Consed software (Russell 2018). Sequencing data and genome characteristics are presented in Table 1.
Bacteriophage genome annotations were completed using DNA Master v5.23.6 (https://phagesdb.org/DNAMaster/) embedded with Glimmer v3.02 (Delcher et al. 2007) and GeneMark v2.5p (Besemer and Borodovsky 2005). Gene start sites were refined using Starterator v1.2 (http://phages.wustl.edu/starterator/). To identify putative functions of protein-coding genes, several comparative tools were used, all with default parameters for all software. These included Phamerator (Cresawn et al. 2011) with the Actino_draft database v578; BLAST, using the Actinobacteriophage and NCBI non-redundant databases (Altschul et al. 1990); and HHPRED, using the PDB_mmCIF70, Pfam v.36, and NCBI Conserved Domains databases (Söding et al. 2005). Aragorn v1.2.41 (Laslett and Canback 2004) or tRNAscanSE v.2.0 (Lowe and Chan 2016) were used to identify tRNAs.
Andrew has a genome of 38,802 bp, with a GC content of 65.5%. RedFox is 38728 bp long, with a 66.1% GC content. Both have 3′ 12-base single-stranded sticky ends. The GC content is typical of other Arthrobacter bacteriophages, which range from 45.1% to 68.5% (Klyczek et al. 2018; Russell and Hatfull 2017). These two bacteriophages were sorted into cluster AS and subcluster AS3 based on gene content similarity (GCS) of at least 35% to bacteriophages in the Actinobacteriophage database (Russell and Hatfull 2017; Pope et al. 2017).
Genome annotation revealed that Andrew contains 72 predicted genes, 36 of which were assigned putative functions, whereas RedFox contains 71 predicted genes, 40 of which were assigned putative functions (Table 1). Neither genome was found to contain tRNAs genes (Table 1). Both Andrew and RedFox demonstrated high GCS of 83.1% and shared highly conserved genome architectures characteristic of the AS3 subcluster (Glaser et al. 2026). Both genomes displayed a modular structure containing conserved regions associated with DNA packaging, virion structure and assembly, lysogeny, and DNA replication (Glaser et al. 2026). Despite this overall conservation, several regions display greater genomic divergence when compared with AS3 cluster phages, including gene 25 in the central region of the Andrew genome and genes 64, 65 and 66 near the end of Andrew genome. These genes lack homologs in the actinobacteriophage database, to date, adding to the diversity of gene sequences uncovered for actinobacteriophages through the isolation and characterization of novel phages.
GenBank accession numbers for Andrew and RedFox are MH834595 and OR195049). SRA accession numbers for Andrew and RedFox are SRX31241826 and SRX20165779.
","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":"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":"Freifelder D. 1983. Molecular biology: a comprehensive introduction to prokaryotes and eukaryotes. Jones and Bartlett. ISBN 10: 086720012X / ISBN 13: 9780867200126
","pubmedId":"","doi":""},{"reference":"Hatfull GF, Dedrick RM, Schooley RT. 2022. Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine 73: 197-211.
","pubmedId":"","doi":"10.1146/annurev-med-080219-122208"},{"reference":"Hibstu Z, Belew H, Akelew Y, Mengist HM. 2022. Phage Therapy: A Different Approach to Fight Bacterial Infections. Biologics: Targets and Therapy Volume 16: 173-186.
","pubmedId":"","doi":"10.2147/BTT.S381237"},{"reference":"Imran A, Shehzadi U, Islam F, Afzaal M, Ali R, Ali YA, et al., Rasool. 2023. Bacteriophages and food safety: An updated overview. Food Science & Nutrition 11: 3621-3630.
","pubmedId":"","doi":"10.1002/fsn3.3360"},{"reference":"Klyczek KK, Jacobs-Sera D, Adair TL, Adams SD, Ball SL, Benjamin RC, et al., Hatfull. 2018. Complete Genome Sequences of 44\n Arthrobacter\n Phages. Genome Announcements 6: 10.1128/genomea.01474-17.
","pubmedId":"","doi":"10.1128/genomea.01474-17"},{"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, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.
","pubmedId":"","doi":"10.1093/nar/gkw413"},{"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":"Rinehart CA, Gaffney BL, Smith JR, Wood JD. 2016. PECAAN: Phage Evidence Collection and Annotation Network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY.
","pubmedId":"","doi":""},{"reference":"Rivka Glaser; Porter, M.; Gibb, B.; Vega, N.; Swerdlow, S.; Klyczek, K.; Denise L Monti (2026). Subcluster AS3 Annotation Report. HHMI Science Education Alliance (SEA) Faculty Group, (Version 2.0). QUBES Educational Resources. doi:10.25334/QB6X-VH05
","pubmedId":"","doi":"10.25334/QB6X-VH05"},{"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 DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"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 Analysis of Two Arthrobacter Bacteriophages, Andrew and RedFox, from Subcluster AS3 Isolated from Soil in New York and Pennsylvania
","reviews":[],"curatorReviews":[]},{"id":"1d38d7f7-a936-4d26-acb2-0b4f58fcbaff","decision":"publish","abstract":"Novel bacteriophages Andrew and RedFox were isolated from soil samples collected in New York and Pennsylvania and propagated using Arthrobacter globiformis B-2979. Both phages are assigned to actinobacteriophage subcluster AS3 based on gene content. Although these phages exhibit conserved genomic content characteristics of the AS3 subcluster, there are notable differences. In particular, phage Andrew gene 25 in the central region of the genome and genes 64, 65, and 66 near the end of the genome lack homologs in cluster AS3 or in the actinobacteriophage database more broadly.
","acknowledgements":"We would like to acknowledge John H. Martinson Honors College, Rowan University College of Science and Mathematics, and Dr. Stephen Bentivenga for their continued support.
We would like to acknowledge the University of Pittsburgh and Marist University SEA-PHAGES faculty and students for the discovery of the Andrew and RedFox bacteriophages and the annotation of the bacteriophages’ genomes.
We would also like to acknowledge Dr. Vic Sivinathan and Dr. Graham Hatfull from HHMI’s SEA-PHAGES program.
","authors":[{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"baldwi87@rowan.edu","firstName":"Paige","lastName":"Baldwin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Megan.Dennis@marist.edu","firstName":"Megan","lastName":"Dennis ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikni78@rowan.edu","firstName":"Niran","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"isikha49@rowan.edu","firstName":"Hanan","lastName":" Isik ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"djs@pitt.edu","firstName":"Deborah","lastName":"Jacobs-Sera ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Juliana.Magna1@marist.edu","firstName":"Juliana","lastName":"Magna ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["investigation","writing_originalDraft"],"email":"mazaka27@rowan.edu","firstName":"Mia","lastName":"Mazakas ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Marist University, Town of Poughkeepsie, NY, United States"],"departments":["Biology"],"credit":["investigation"],"email":"Gabrielle.Rice1@marist.edu","firstName":"Gabriella","lastName":"Rice","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"sswerdlow@pitt.edu","firstName":"Sarah","lastName":"Swerdlow ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["University of Pittsburgh, Pittsburgh, PA, United States"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"mcu7@pitt.edu","firstName":"Megan","lastName":"Ulbrich ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization"],"email":"farberm@rowan.edu","firstName":"Matthew ","lastName":"Farber","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Rowan University, Glassboro, NJ, United States"],"departments":["Biological & Biomedical Sciences"],"credit":["supervision","conceptualization","validation","investigation","writing_reviewEditing"],"email":"bogush@rowan.edu","firstName":"Marina","lastName":"Bogush ","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/17d50d2190b1f1631c6528eb797465bb.csv"},"extendedData":[],"funding":"This project has been supported by the Department of Biological and Biomedical Sciences, College of Science and Mathematics, Rowan University, Glassboro, New Jersey
","image":{"url":null},"imageCaption":"Sequencing data and genome characteristics
","imageTitle":"Bacteriophages Andrew and RedFox
","methods":"","reagents":"","patternDescription":"Bacteriophages provide a means to modulate bacterial populations and therefore have practical applications in medicine and industry, including the treatment of bacterial infections (Hibstu et al. 2022; Hatfull et al. 2022) and the preservation of food (Imran et al. 2023). Given their abundance and diversity, the isolation and characterization of novel bacteriophages promise to advance their application. Here, we described the isolation and characterization of two bacteriophages using Arthrobacter globiformis, a soil bacterium that is both widely distributed and ecologically significant (Klyczek et al., 2018).
Bacteriophages Andrew and RedFox were isolated by enrichment (Zorawik et al. 2024) from soil samples collected in mildly wet and nutrient-rich environments in Pittsburgh, Pennsylvania (GPS coordinate: 40.445472 N, 79.998708 W) and Poughkeepsie, New York (GPS coordinate: 41.7203 N, 73.9356 W), respectively. Andrew and RedFox soil samples were suspended in peptone-yeast extract-calcium (PYCa) medium and shaken at 250 rpm for 1 and 1.5 hours, respectively. The suspensions were centrifuged, filtered through a 0.22-μm filter, and the filtrate inoculated with Arthrobacter globiformis B-2979 for 48 hours at 30°C. The enriched cultures were then filtered again, and 10 μL of each filtrate was plated in PYCa top agar with A. globiformis and incubated at 30°C for 48 hours. Phages were purified through three successive rounds of plating for isolated plaques. Both Andrew and RedFox produced clear plaques.
Transmission electron microscopy with 1% uranyl acetate staining for Andrew or UranyLess staining for RedFox revealed siphovirus morphologies characterized by icosahedral capsids and flexible tails. The corresponding plaque and transmission electron microscopy (TEM) images are available through the PhagesDB entries for Andrew and RedFox, respectively (Russell and Hatfull 2017). DNA was extracted from bacteriophage lysates using the Promega DNA Wizard Cleanup Kit. Sequencing libraries were prepared using the NEB Ultra II FS DNA library kit and run on the Illumina MiSeq platform (v3 reagents), yielding 150-base single-end reads that were assembled into genomes using Newbler and Consed software (Russell 2018). Sequencing data and genome characteristics are presented in Table 1.
Bacteriophage genome annotations were completed using DNA Master v5.23.6 (https://phagesdb.org/DNAMaster/) embedded with Glimmer v3.02 (Delcher et al. 2007) and GeneMark v2.5p (Besemer and Borodovsky 2005). Gene start sites were refined using Starterator v1.2 (http://phages.wustl.edu/starterator/). To identify putative functions of protein-coding genes, several comparative tools were used, all with default parameters for all software. These included Phamerator (Cresawn et al. 2011) with the Actino_draft database v578; BLAST, using the Actinobacteriophage and NCBI non-redundant databases (Altschul et al. 1990); and HHPRED, using the PDB_mmCIF70, Pfam v.36, and NCBI Conserved Domains databases (Söding et al. 2005). Aragorn v1.2.41 (Laslett and Canback 2004) or tRNAscanSE v.2.0 (Lowe and Chan 2016) were used to identify tRNAs.
Andrew has a genome of 38,802 bp, with a GC content of 65.5%. RedFox is 38728 bp long, with a 66.1% GC content. Both have 3′ 12-base single-stranded sticky ends. The GC content is typical of other Arthrobacter bacteriophages, which range from 45.1% to 68.5% (Klyczek et al. 2018; Russell and Hatfull 2017). These two bacteriophages were sorted into cluster AS and subcluster AS3 based on gene content similarity (GCS) of at least 35% to bacteriophages in the Actinobacteriophage database (Russell and Hatfull 2017; Pope et al. 2017).
Genome annotation revealed that Andrew contains 72 predicted genes, 36 of which were assigned putative functions, whereas RedFox contains 71 predicted genes, 40 of which were assigned putative functions (Table 1). Neither genome was found to contain tRNAs genes (Table 1). Both Andrew and RedFox demonstrated high GCS of 83.1% and shared highly conserved genome architectures characteristic of the AS3 subcluster (Glaser et al. 2026). Both genomes displayed a modular structure containing conserved regions associated with DNA packaging, virion structure and assembly, lysogeny, and DNA replication (Glaser et al. 2026). Despite this overall conservation, several regions display greater genomic divergence when compared with AS3 cluster phages, including gene 25 in the central region of the Andrew genome and genes 64, 65 and 66 near the end of Andrew genome. These genes lack homologs in the actinobacteriophage database, to date, adding to the diversity of gene sequences uncovered for actinobacteriophages through the isolation and characterization of novel phages.
GenBank accession numbers for Andrew and RedFox are MH834595 and OR195049). SRA accession numbers for Andrew and RedFox are SRX31241826 and SRX20165779.
","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":"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":"Freifelder D. 1983. Molecular biology: a comprehensive introduction to prokaryotes and eukaryotes. Jones and Bartlett. ISBN 10: 086720012X / ISBN 13: 9780867200126
","pubmedId":"","doi":""},{"reference":"Hatfull GF, Dedrick RM, Schooley RT. 2022. Phage Therapy for Antibiotic-Resistant Bacterial Infections. Annual Review of Medicine 73: 197-211.
","pubmedId":"","doi":"10.1146/annurev-med-080219-122208"},{"reference":"Hibstu Z, Belew H, Akelew Y, Mengist HM. 2022. Phage Therapy: A Different Approach to Fight Bacterial Infections. Biologics: Targets and Therapy Volume 16: 173-186.
","pubmedId":"","doi":"10.2147/BTT.S381237"},{"reference":"Imran A, Shehzadi U, Islam F, Afzaal M, Ali R, Ali YA, et al., Rasool. 2023. Bacteriophages and food safety: An updated overview. Food Science & Nutrition 11: 3621-3630.
","pubmedId":"","doi":"10.1002/fsn3.3360"},{"reference":"Klyczek KK, Jacobs-Sera D, Adair TL, Adams SD, Ball SL, Benjamin RC, et al., Hatfull. 2018. Complete Genome Sequences of 44\n Arthrobacter\n Phages. Genome Announcements 6: 10.1128/genomea.01474-17.
","pubmedId":"","doi":"10.1128/genomea.01474-17"},{"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, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.
","pubmedId":"","doi":"10.1093/nar/gkw413"},{"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":"Rinehart CA, Gaffney BL, Smith JR, Wood JD. 2016. PECAAN: Phage Evidence Collection and Annotation Network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY.
","pubmedId":"","doi":""},{"reference":"Rivka Glaser; Porter, M.; Gibb, B.; Vega, N.; Swerdlow, S.; Klyczek, K.; Denise L Monti (2026). Subcluster AS3 Annotation Report. HHMI Science Education Alliance (SEA) Faculty Group, (Version 2.0). QUBES Educational Resources. doi:10.25334/QB6X-VH05
","pubmedId":"","doi":"10.25334/QB6X-VH05"},{"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 DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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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