Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson
UNG Honors Program
Georgia Microscopy facility Mary Ard UGA SEA-PHAGES program. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/37376fe59665aa2ac13be8a25c1b4a13.png"},"imageCaption":"The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Structure prediction of ORF 117 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Transmission electron micrograph (TEM) of Lethe
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered and the filtrate plated in 7H9 to agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are small sized, clear, and rounded with defined edges. A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phophotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1).
Phage genomic DNA was isolated from Lethe’s lysate using a Wizard DNA extraction kit (Promega) per manufacturer instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017) .
Lethe’s genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 and DNA Master v4.2.1.11 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1B), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages, which has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection by these subset of cluster C1 phages (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977nd Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J. 2009 Apr 8;28(7):821-9. doi: 10.1038/emboj.2009.36. Epub 2009 Feb 19. Erratum in: EMBO J. 2012 Aug 15;31(16):3507. PMID: 19229296; PMCID: PMC2670864
","pubmedId":"","doi":""},{"reference":"Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol, 215(3), 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
","pubmedId":"","doi":""},{"reference":"Besemer, J., & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res, 33(Web Server issue), W451-454. https://doi.org/10.1093/nar/gki487
","pubmedId":"","doi":""},{"reference":"Chan P, Lin B, Mak A, Lowe T. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes, Nucleic Acids Research, 49,16, 9077–9096, https://doi.org/10.1093/nar/gkab688
","pubmedId":"","doi":""},{"reference":"Chen, Y., Yu, P., Luo, J., & Jiang, Y. (2003). Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome, 14(12), 859-865. https://doi.org/10.1007/s00335-003-2296-6
","pubmedId":"","doi":""},{"reference":"Cresawn, S. G., Bogel, M., Day, N., Jacobs-Sera, D., Hendrix, R. W., & Hatfull, G. F. (2011). Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics, 12, 395. https://doi.org/10.1186/1471-2105-12-395
","pubmedId":"","doi":""},{"reference":"Delcher, A. L., Bratke, K. A., Powers, E. C., & Salzberg, S. L. (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics, 23(6), 673-679. https://doi.org/10.1093/bioinformatics/btm009
","pubmedId":"","doi":""},{"reference":"Heller, D. M., Sivanathan, V., Asai, D. J., & Hatfull, G. F. (2024). Sea-phages and sea-genes: Advancing Virology and Science Education. Annual Review of Virology, 11(1), 1–20. https://doi.org/10.1146/annurev-virology-113023-110757
","pubmedId":"","doi":""},{"reference":"Laslett, D., & Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res, 32(1), 11-16. https://doi.org/10.1093/nar/gkh152
","pubmedId":"","doi":""},{"reference":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, ... Hatfull GF. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One. 2011 Jan 27;6(1):e16329. doi: 10.1371/journal.pone.0016329. PMID: 21298013; PMCID: PMC3029335.
","pubmedId":"","doi":""},{"reference":"Pope, W. H., & Jacobs-Sera, D. (2018). Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods Mol Biol, 1681, 217-229. https://doi.org/10.1007/978-1-4939-7343-9_16
","pubmedId":"","doi":""},{"reference":"Russell DA, H. G. (2017). the actinobacteriophage database. Bioinformatics, 1(5), 784-786.
","pubmedId":"","doi":""},{"reference":"Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40. https://doi.org/10.1186/1471-2105-9-40
","pubmedId":"","doi":""},{"reference":"Zimmermann, L., Stephens, A., Nam, S. Z., Rau, D., Kubler, J., Lozajic, M., Gabler, F., Soding, J., Lupas, A. N., & Alva, V. (2018). A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol, 430(15), 2237-2243.
","pubmedId":"","doi":""},{"reference":"Zorawik, M., Jacobs-Sera, D., Freise, A. C., Sea, P., & Reddi, K. (2024). Isolation of Bacteriophages on Actinobacteria Hosts. Methods Mol Biol, 2793, 273-298. https://doi.org/10.1007/978-1-0716-3798-2_17
","pubmedId":"","doi":""}],"title":"Genome of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[],"curatorReviews":[]},{"id":"51eae6da-39c3-4ec5-aace-d3da7d2b181b","decision":"revise","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/37376fe59665aa2ac13be8a25c1b4a13.png"},"imageCaption":"The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Transmission electron micrograph (TEM) of Lethe
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated in 7H9 to agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are small sized, clear, and rounded with defined edges. A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phophotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1).
Phage genomic DNA was isolated from Lethe’s lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017) .
Lethe’s genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 and DNA Master v4.2.1.11 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1B), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection by these subset of cluster C1 phages (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977nd Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J. 2009 Apr 8;28(7):821-9. doi: 10.1038/emboj.2009.36. Epub 2009 Feb 19. Erratum in: EMBO J. 2012 Aug 15;31(16):3507. PMID: 19229296; PMCID: PMC2670864
","pubmedId":"","doi":""},{"reference":"Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol, 215(3), 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
","pubmedId":"","doi":""},{"reference":"Besemer, J., & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res, 33(Web Server issue), W451-454. https://doi.org/10.1093/nar/gki487
","pubmedId":"","doi":""},{"reference":"Chan P, Lin B, Mak A, Lowe T. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes, Nucleic Acids Research, 49,16, 9077–9096, https://doi.org/10.1093/nar/gkab688
","pubmedId":"","doi":""},{"reference":"Chen, Y., Yu, P., Luo, J., & Jiang, Y. (2003). Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome, 14(12), 859-865. https://doi.org/10.1007/s00335-003-2296-6
","pubmedId":"","doi":""},{"reference":"Cresawn, S. G., Bogel, M., Day, N., Jacobs-Sera, D., Hendrix, R. W., & Hatfull, G. F. (2011). Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics, 12, 395. https://doi.org/10.1186/1471-2105-12-395
","pubmedId":"","doi":""},{"reference":"Delcher, A. L., Bratke, K. A., Powers, E. C., & Salzberg, S. L. (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics, 23(6), 673-679. https://doi.org/10.1093/bioinformatics/btm009
","pubmedId":"","doi":""},{"reference":"Heller, D. M., Sivanathan, V., Asai, D. J., & Hatfull, G. F. (2024). Sea-phages and sea-genes: Advancing Virology and Science Education. Annual Review of Virology, 11(1), 1–20. https://doi.org/10.1146/annurev-virology-113023-110757
","pubmedId":"","doi":""},{"reference":"Laslett, D., & Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res, 32(1), 11-16. https://doi.org/10.1093/nar/gkh152
","pubmedId":"","doi":""},{"reference":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, ... Hatfull GF. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One. 2011 Jan 27;6(1):e16329. doi: 10.1371/journal.pone.0016329. PMID: 21298013; PMCID: PMC3029335.
","pubmedId":"","doi":""},{"reference":"Pope, W. H., & Jacobs-Sera, D. (2018). Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods Mol Biol, 1681, 217-229. https://doi.org/10.1007/978-1-4939-7343-9_16
","pubmedId":"","doi":""},{"reference":"Russell DA, H. G. (2017). the actinobacteriophage database. Bioinformatics, 1(5), 784-786.
","pubmedId":"","doi":""},{"reference":"Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40. https://doi.org/10.1186/1471-2105-9-40
","pubmedId":"","doi":""},{"reference":"Zimmermann, L., Stephens, A., Nam, S. Z., Rau, D., Kubler, J., Lozajic, M., Gabler, F., Soding, J., Lupas, A. N., & Alva, V. (2018). A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol, 430(15), 2237-2243.
","pubmedId":"","doi":""},{"reference":"Zorawik, M., Jacobs-Sera, D., Freise, A. C., Sea, P., & Reddi, K. (2024). Isolation of Bacteriophages on Actinobacteria Hosts. Methods Mol Biol, 2793, 273-298. https://doi.org/10.1007/978-1-0716-3798-2_17
","pubmedId":"","doi":""}],"title":"Genome of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[{"reviewer":{"displayName":"Sara Tolsma"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Marie Fogarty"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"bbd116c5-c7d1-441d-8d05-41adce938865","decision":"accept","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/55a8c80b5aececf2238e0a173adbf17e.png"},"imageCaption":"A. Transmission electron micrograph (TEM) of Lethe. The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Representative plaques produced by Lethe are turbid and 1-5 mm in size. C. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Structure of phage particles, plaques, and protein
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated in 7H9 to agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are 1-5 mm in diameter, turbid, and rounded with defined edges. A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phophotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1).
Phage genomic DNA was isolated from Lethe's lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017).
Lethe's genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 (Rinehart CA, Gaffney BL, et al. 2016). and DNA Master V5.23.6 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1B), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977 and Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J. 2009 Apr 8;28(7):821-9. doi: 10.1038/emboj.2009.36. Epub 2009 Feb 19. Erratum in: EMBO J. 2012 Aug 15;31(16):3507. PMID: 19229296; PMCID: PMC2670864
","pubmedId":"","doi":""},{"reference":"Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol, 215(3), 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
","pubmedId":"","doi":""},{"reference":"Besemer, J., & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res, 33(Web Server issue), W451-454. https://doi.org/10.1093/nar/gki487
","pubmedId":"","doi":""},{"reference":"Chan P, Lin B, Mak A, Lowe T. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes, Nucleic Acids Research, 49,16, 9077–9096, https://doi.org/10.1093/nar/gkab688
","pubmedId":"","doi":""},{"reference":"Chen, Y., Yu, P., Luo, J., & Jiang, Y. (2003). Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome, 14(12), 859-865. https://doi.org/10.1007/s00335-003-2296-6
","pubmedId":"","doi":""},{"reference":"Cresawn, S. G., Bogel, M., Day, N., Jacobs-Sera, D., Hendrix, R. W., & Hatfull, G. F. (2011). Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics, 12, 395. https://doi.org/10.1186/1471-2105-12-395
","pubmedId":"","doi":""},{"reference":"Delcher, A. L., Bratke, K. A., Powers, E. C., & Salzberg, S. L. (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics, 23(6), 673-679. https://doi.org/10.1093/bioinformatics/btm009
","pubmedId":"","doi":""},{"reference":"Heller, D. M., Sivanathan, V., Asai, D. J., & Hatfull, G. F. (2024). Sea-phages and sea-genes: Advancing Virology and Science Education. Annual Review of Virology, 11(1), 1–20. https://doi.org/10.1146/annurev-virology-113023-110757
","pubmedId":"","doi":""},{"reference":"Laslett, D., & Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res, 32(1), 11-16. https://doi.org/10.1093/nar/gkh152
","pubmedId":"","doi":""},{"reference":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, ... Hatfull GF. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One. 2011 Jan 27;6(1):e16329. doi: 10.1371/journal.pone.0016329. PMID: 21298013; PMCID: PMC3029335.
","pubmedId":"","doi":""},{"reference":"Pope, W. H., & Jacobs-Sera, D. (2018). Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods Mol Biol, 1681, 217-229. https://doi.org/10.1007/978-1-4939-7343-9_16
","pubmedId":"","doi":""},{"reference":"Russell DA, H. G. (2017). the actinobacteriophage database. Bioinformatics, 1(5), 784-786.
","pubmedId":"","doi":""},{"reference":"Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40. https://doi.org/10.1186/1471-2105-9-40
","pubmedId":"","doi":""},{"reference":"Zimmermann, L., Stephens, A., Nam, S. Z., Rau, D., Kubler, J., Lozajic, M., Gabler, F., Soding, J., Lupas, A. N., & Alva, V. (2018). A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol, 430(15), 2237-2243.
","pubmedId":"","doi":""},{"reference":"Zorawik, M., Jacobs-Sera, D., Freise, A. C., Sea, P., & Reddi, K. (2024). Isolation of Bacteriophages on Actinobacteria Hosts. Methods Mol Biol, 2793, 273-298. https://doi.org/10.1007/978-1-0716-3798-2_17
","pubmedId":"","doi":""},{"reference":"Rinehart, C.A., Gaffney, B.L., Smith, J.R. and Wood, J.D., 2016. PECAAN: bacteriophage evidence collection and annotation network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY
","pubmedId":"","doi":""}],"title":"Genome of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[],"curatorReviews":[]},{"id":"8fd1f398-b9f3-4a4c-83f9-46dd5c08ab39","decision":"edit","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/f5a9866ed2eb7f36e1b32fe1bda8f180.png"},"imageCaption":"A. Transmission electron micrograph (TEM) of Lethe. The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Representative plaques produced by Lethe are turbid and 1-5 mm in size. C. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Structure of phage particles, plaques, and protein
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated on 7H10 agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are 1-5 mm in diameter, turbid, and rounded with defined edges (Fig 1B). A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phosphotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1A).
Phage genomic DNA was isolated from Lethe's lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017).
Lethe's genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 (Rinehart CA, Gaffney BL, et al. 2016). and DNA Master V5.23.6 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1C), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977 and Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J. 2009 Apr 8;28(7):821-9. doi: 10.1038/emboj.2009.36. Epub 2009 Feb 19. Erratum in: EMBO J. 2012 Aug 15;31(16):3507. PMID: 19229296; PMCID: PMC2670864
","pubmedId":"","doi":""},{"reference":"Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol, 215(3), 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
","pubmedId":"","doi":""},{"reference":"Besemer, J., & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res, 33(Web Server issue), W451-454. https://doi.org/10.1093/nar/gki487
","pubmedId":"","doi":""},{"reference":"Chan P, Lin B, Mak A, Lowe T. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes, Nucleic Acids Research, 49,16, 9077–9096, https://doi.org/10.1093/nar/gkab688
","pubmedId":"","doi":""},{"reference":"Chen, Y., Yu, P., Luo, J., & Jiang, Y. (2003). Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome, 14(12), 859-865. https://doi.org/10.1007/s00335-003-2296-6
","pubmedId":"","doi":""},{"reference":"Cresawn, S. G., Bogel, M., Day, N., Jacobs-Sera, D., Hendrix, R. W., & Hatfull, G. F. (2011). Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics, 12, 395. https://doi.org/10.1186/1471-2105-12-395
","pubmedId":"","doi":""},{"reference":"Delcher, A. L., Bratke, K. A., Powers, E. C., & Salzberg, S. L. (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics, 23(6), 673-679. https://doi.org/10.1093/bioinformatics/btm009
","pubmedId":"","doi":""},{"reference":"Heller, D. M., Sivanathan, V., Asai, D. J., & Hatfull, G. F. (2024). Sea-phages and sea-genes: Advancing Virology and Science Education. Annual Review of Virology, 11(1), 1–20. https://doi.org/10.1146/annurev-virology-113023-110757
","pubmedId":"","doi":""},{"reference":"Laslett, D., & Canback, B. (2004). ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res, 32(1), 11-16. https://doi.org/10.1093/nar/gkh152
","pubmedId":"","doi":""},{"reference":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, ... Hatfull GF. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One. 2011 Jan 27;6(1):e16329. doi: 10.1371/journal.pone.0016329. PMID: 21298013; PMCID: PMC3029335.
","pubmedId":"","doi":""},{"reference":"Pope, W. H., & Jacobs-Sera, D. (2018). Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods Mol Biol, 1681, 217-229. https://doi.org/10.1007/978-1-4939-7343-9_16
","pubmedId":"","doi":""},{"reference":"Russell DA, H. G. (2017). the actinobacteriophage database. Bioinformatics, 1(5), 784-786.
","pubmedId":"","doi":""},{"reference":"Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40. https://doi.org/10.1186/1471-2105-9-40
","pubmedId":"","doi":""},{"reference":"Zimmermann, L., Stephens, A., Nam, S. Z., Rau, D., Kubler, J., Lozajic, M., Gabler, F., Soding, J., Lupas, A. N., & Alva, V. (2018). A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol, 430(15), 2237-2243.
","pubmedId":"","doi":""},{"reference":"Zorawik, M., Jacobs-Sera, D., Freise, A. C., Sea, P., & Reddi, K. (2024). Isolation of Bacteriophages on Actinobacteria Hosts. Methods Mol Biol, 2793, 273-298. https://doi.org/10.1007/978-1-0716-3798-2_17
","pubmedId":"","doi":""},{"reference":"Rinehart, C.A., Gaffney, B.L., Smith, J.R. and Wood, J.D., 2016. PECAAN: bacteriophage evidence collection and annotation network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY
","pubmedId":"","doi":""}],"title":"Genome of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[],"curatorReviews":[]},{"id":"3abd8d1d-135d-422d-9335-a369c92a3f96","decision":"accept","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/f5a9866ed2eb7f36e1b32fe1bda8f180.png"},"imageCaption":"A. Transmission electron micrograph (TEM) of Lethe. The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Representative plaques produced by Lethe are turbid and 1-5 mm in size. C. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Structure of phage particles, plaques, and protein
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated on 7H10 agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are 1-5 mm in diameter, turbid, and rounded with defined edges (Fig 1B). A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phosphotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1A).
Phage genomic DNA was isolated from Lethe's lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017).
Lethe's genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 (Rinehart CA, Gaffney BL, et al. 2016). and DNA Master V5.23.6 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1C), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977 and Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. 2009. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J 28(7): 821-9.
","pubmedId":"19229296","doi":""},{"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":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Research 49: 9077-9096.
","pubmedId":"","doi":"10.1093/nar/gkab688"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"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":"Heller DM, Sivanathan V, Asai DJ, Hatfull GF. 2024. SEA-PHAGES and SEA-GENES: Advancing Virology and Science Education. Annual Review of Virology 11: 1-20.
","pubmedId":"","doi":"10.1146/annurev-virology-113023-110757"},{"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":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, et al., Hatfull GF. 2011. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One 6(1): e16329.
","pubmedId":"21298013","doi":""},{"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":"Rinehart, C.A., Gaffney, B.L., Smith, J.R. and Wood, J.D., 2016. PECAAN: bacteriophage evidence collection and annotation network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33(5): 784-786.
","pubmedId":"28365761","doi":""},{"reference":"Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9: 10.1186/1471-2105-9-40.
","pubmedId":"","doi":"10.1186/1471-2105-9-40"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al., Alva V. 2018. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol 430(15): 2237-2243.
","pubmedId":"29258817","doi":""},{"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 of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[],"curatorReviews":[]},{"id":"acb1f7db-16a1-4629-a3f8-26a43315d715","decision":"edit","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/f5a9866ed2eb7f36e1b32fe1bda8f180.png"},"imageCaption":"A. Transmission electron micrograph (TEM) of Lethe. The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Representative plaques produced by Lethe are turbid and 1-5 mm in size. C. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Structure of phage particles, plaques, and protein
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated on 7H10 agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are 1-5 mm in diameter, turbid, and rounded with defined edges (Fig 1B). A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phosphotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1A).
Phage genomic DNA was isolated from Lethe's lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017).
Lethe's genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 (Rinehart CA, Gaffney BL, et al. 2016). and DNA Master V5.23.6 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1C), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977 and Sequence Read Archive (SRA) No. SRX29990114.
","references":[{"reference":"Aksyuk AA, Leiman PG, Kurochkina LP, Shneider MM, Kostyuchenko VA, Mesyanzhinov VV, Rossmann MG. 2009. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J 28(7): 821-9.
","pubmedId":"19229296","doi":""},{"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":"Chan PP, Lin BY, Mak AJ, Lowe TM. 2021. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Research 49: 9077-9096.
","pubmedId":"","doi":"10.1093/nar/gkab688"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"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":"Heller DM, Sivanathan V, Asai DJ, Hatfull GF. 2024. SEA-PHAGES and SEA-GENES: Advancing Virology and Science Education. Annual Review of Virology 11: 1-20.
","pubmedId":"","doi":"10.1146/annurev-virology-113023-110757"},{"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":"Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, et al., Hatfull GF. 2011. Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution. PLoS One 6(1): e16329.
","pubmedId":"21298013","doi":""},{"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":"Rinehart, C.A., Gaffney, B.L., Smith, J.R. and Wood, J.D., 2016. PECAAN: bacteriophage evidence collection and annotation network user guide. Western Kentucky University Bioinformatics and Information Science Center, Bowling Green, KY
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33(5): 784-786.
","pubmedId":"28365761","doi":""},{"reference":"Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9: 10.1186/1471-2105-9-40.
","pubmedId":"","doi":"10.1186/1471-2105-9-40"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al., Alva V. 2018. A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol 430(15): 2237-2243.
","pubmedId":"29258817","doi":""},{"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 of Myoviridae Phage Lethe Isolated In Northern Georgia
","reviews":[],"curatorReviews":[]},{"id":"2956d579-8d63-49ce-91ad-d9a317f23254","decision":"publish","abstract":"Lethe, a predicted lytic bacteriophage with myovirus morphology, was isolated using Mycobacterium smegmatis mc²155 in North Georgia. Lethe has a genome of 155,828 base pairs with 230 predicted ORFs, 34 tRNAs, 1 tmRNA, and 64.70% GC content. Based on gene content, Lethe is assigned to actinobacteriophage cluster C1, sharing up to 99.5% nucleotide identity with members of this cluster.
","acknowledgements":"TAs Audrey Nesbit and Nathan Simpson devoted countless hours preparing samples and aiding in the lab, without which none of this work could be done. The UNG Honors Program provided support and printing abilities. The Georgia Microscopy facility Mary Ard at UGA provided grids, stain, and expertise to image Lethe. This work used the JEOL 2100PLUS microscope housed in UGA's Georgia Electron Microscopy core facility that was acquired with funding from the National Institutes of Health through grant 1S10OD034282-01. The SEA-PHAGES program provided protocols and technical support.
","authors":[{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration","formalAnalysis","investigation"],"email":"PMALEX0691@ung.edu","firstName":"Paige","lastName":"Alexander","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"MEBLAI6106@ung.edu","firstName":"Maggie","lastName":"Blair","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EKBOYE3484@ung.edu","firstName":"Emma ","lastName":"Boyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"ECBUMG3127@ung.edu","firstName":"Lavender","lastName":"Bumgarner","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"GEGROG2775@ung.edu","firstName":"Gabrielle","lastName":"Grogan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":[""],"credit":["dataCuration"],"email":"EALESP5196@ung.edu","firstName":"Ella","lastName":"Lesperance","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of North Georgia, Dahlonega, GA, US"],"departments":["Biology"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"aekanak@ung.edu","firstName":"Alison ","lastName":"Kanak","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9053-250X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"N/A
","image":{"url":"https://portal.micropublication.org/uploads/f5a9866ed2eb7f36e1b32fe1bda8f180.png"},"imageCaption":"A. Transmission electron micrograph (TEM) of Lethe. The TEM was obtained using a J JEOL 2100PLUS (JOEL, Inc., Tokyo, Japan) at the University of Georgia Electron Microscope laboratory. B. Representative plaques produced by Lethe are turbid and 1-5 mm in size. C. Structure prediction of ORF 125 using I-TASSER includes multiple alpha helices (in magenta) and beta sheets (in orange).
","imageTitle":"Structure of phage particles, plaques, and protein
","methods":"","reagents":"","patternDescription":"The study of bacteriophage, viruses that prey on bacterial hosts, is increasingly important due to their use as an alternative to antibiotics. Complementing these efforts is the SEA-PHAGES program that mobilizes thousands of undergraduate students in isolating and characterizing novel bacteriophages (Heller et al, 2024).
Lethe was isolated from an environmental sample collected at Lake Zwerner, which is in northeast downtown Dahlonega, GA (34.55437°N, 83.96678°W). The sample was fine, silt like, watery, light brown soil from the bottom of the lake. After collection, the sample was filtered then suspended in Middlebrook 7H9 liquid medium and the suspension was inoculated with Mycobacterium smegmatis mc2 155 and incubated with shaking overnight at 37°C. The resulting culture was filtered with a 0.22µm filter and the filtrate plated on 7H10 agar with M. smegmatis in a standard plaque assay (Zorawik et al., 2024). Plates were incubated at 37°C for 48 hours. After four rounds of picking plaques and plating, a clonal population of Lethe was found to form plaques that are 1-5 mm in diameter, turbid, and rounded with defined edges (Fig 1B). A high titer lysate of Lethe was prepared and used for electron microscopy and genomic DNA extraction. Phosphotungstic acid (1 %) and a parafilm drop method was used to stain and mount the phage sample for negative stain transmission electron microscopy, which revealed Lethe to possess a myovirus morphology with a capsid measuring 69.12 nm in diameter as well as a tail measuring 80.96 nm in length as measured using eleif.net/photomeasure (n=1; Figure 1A).
Phage genomic DNA was isolated from Lethe's lysate using a Wizard DNA extraction kit (Promega) per standard Science Education Alliance (SEA) protocol instructions. DNA was prepared for sequencing using the NEB Ultra II Library Kit 9 and sequenced using an Illumina NextSeq 1000 with XLEAP-P1 reagents. The Illumina shotgun sequencing method with an Illumina NextSeq 1000 (XLEAP-P1 kit) was used yielding 2.5 million 100 base reads that resulted in 1529 coverage of an assembled genome. The genome is 155828 bps long with 64.7% GC content and has circularly permuted ends. Lethe is a member of the C1 cluster of Actinobacteriophage due to gene content similarity of at least 35% to phages in the Actinobacteriophage database, phagesdb.org (Russell & Hatfull, 2017; Pope et al, 2017).
Lethe's genome was automatically annotated using Glimmer v3.02 (Delcher et al., 2007) and GeneMark v2.5p (Besemer & Borodovsky, 2005) through PECAAN v20240320 (Rinehart CA, Gaffney BL, et al. 2016). and DNA Master V5.23.6 (Pope & Jacobs-Sera, 2018). The annotation was then manually refined using Starterator v557, Phamerator v557 (Cresawn et al., 2011) using the Actino_draft database, NCBI BLASTp v2.15.0 (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, HHPred v2.08 (Zimmermann et al., 2018) searches against the PDB_mmCIF70, Pfam-v.36, NCBI Conserved Domains databases, tRNAscanSE v2.0 (Chan, et al 2021), Aragorn (Laslett and Canback 2004) TMHMM v.1.0.24 (Chen et al., 2003), TOPCONS v2.0, and I-TASSER (Zhang, 2008). All software was run on the default parameters. Where applicable, hits with E values of 10e-10 or less were considered acceptable. Lethe has 230 predicted open reading frames. Putative functions were predicted for 51 of these, including a predicted tail sheath protein (gp125). Using I-TASSER (Figure 1C), the multiple alpha helices and beta sheets that are key to the structure and functioning of the contractile myoviurs tail can be observed (Akysukk et al., 2009). Lethe also encodes 34 tRNAs and one tmRNA. All but five genes are transcribed unidirectionally. These features are consistent with phages in the C1 cluster, of which there are over 200, to-date. Like other phages in this cluster, Lethe is predicted to be lytic (Hatfull, 2012) and does not encode the immunity repressor of cluster A phages. This has been observed for a small but growing subset of C1 phages and is predicted to provide superinfection immunity to cluster A during lytic infection (Pope et al., 2011). A blastn search of the Lethe genomic sequence against the Actinobacteriophage database reveals C1 phage Iota (Genbank accession no. MK359330.1) as the most genetically similar phage, with 99% percent nucleotide identity across a majority of the genome.
Nucleotide sequence accession numbers
Lethe is available at GenBank with Accession No. PV876977 and Sequence Read Archive (SRA) No. SRX29990114.
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","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome of Myoviridae Phage Lethe Isolated In Northern Georgia
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