Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writingOriginalDraft","writing�ReviewAndEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Computer Sciences"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Chemistry"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writingOriginalDraft","writing�ReviewAndEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Figure: Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Figure: Particle and plaque morphologies for Lucia, Reccee, and Cleatunium
Table: Phage sample locations and genome assembly results
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=6) with a clear center and a turbid edge; Reccee (isolated from an enriched sample) created plaques which were smaller (1.5-2 mm, n=5) and clear, while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia and Reccee exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology.
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208,andPZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no.SRX34536418,SRX34536435, andSRX34536424,respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
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","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[],"curatorReviews":[]},{"id":"0adf74c5-c75c-429c-aca8-f15902448406","decision":"edit","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writingOriginalDraft","writing�ReviewAndEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Computer Sciences"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Chemistry"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing�OriginalDraft","writing�ReviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writingOriginalDraft","writing�ReviewAndEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium. Table: Phage sample locations and genome assembly results
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=6) with a clear center and a turbid edge; Reccee (isolated from an enriched sample) created plaques which were smaller (1.5-2 mm, n=5) and clear, while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia and Reccee exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology.
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208,andPZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no.SRX34536418,SRX34536435, andSRX34536424,respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
","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":"Bendele, M., Cobb, I., and Cresawn, S. Subclusters. Accessed June 7, 2026. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed
","pubmedId":"","doi":""},{"reference":"Bernsel A, Viklund H, Hennerdal A, Elofsson A. 2009. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Research 37: W465-W468.
","pubmedId":"","doi":"10.1093/nar/gkp363"},{"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":"Cobb ID, Cooper K, Bendele MG, Cresawn SG. Accessed June 7, 2026. Observable. https://observablehq.com/@cresawn-labs/cluster-report
","pubmedId":"","doi":""},{"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.
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","pubmedId":"","doi":"10.4014/jmb.2601.01025"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
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","pubmedId":"","doi":"10.1038/s41467-019-13384-y"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
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","pubmedId":"","doi":""},{"reference":"Park N, Cho Y, Kang Y, Kong M. 2026. Characterization of Endolysin LysG77YL from\n Bacillus licheniformis\n -Infecting Bacteriophage G77YL and Application as an Antimicrobial Agent. Journal of Microbiology and Biotechnology 36: 10.4014/jmb.2601.01064.
","pubmedId":"","doi":"10.4014/jmb.2601.01064"},{"reference":"Pardo-Freire M, Bernabéu-Gimeno M, Chan BK, Turner PE, Sánchez-Romero I, Aguilar-Pérez M, Erro-Iribarren M, Domingo-Calap P. 2025. Resolution of acute rejection in a bilateral double-lung transplanted cystic fibrosis patient following phage intervention. ASM Case Reports 1: 10.1128/asmcr.00058-24.
","pubmedId":"","doi":"10.1128/asmcr.00058-24"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
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","pubmedId":"","doi":"10.1128/mBio.01069-17"},{"reference":"Rinehart CA, Gaffney B, Wood JD, Smith S. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Shimamura M, Becken B, Tansmore J, Cristinziano M, Abad L, Dedrick RM, et al., Dousa. 2025. Successful treatment of macrolide-resistant\n Mycobacterium abscessus\n infection using multi-drug regimens including dual β-lactams and phage therapy: case reports in two children. ASM Case Reports 1: 10.1128/asmcr.00087-24.
","pubmedId":"","doi":"10.1128/asmcr.00087-24"},{"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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","pubmedId":"","doi":"10.1016/j.jfp.2025.100557"},{"reference":"Wang C, Niu Y, Yu X, Zhang P, Lu Q, Li Y, et al., Zhang. 2026. Biocontrol of multidrug-resistant Proteus mirabilis on chicken breast, pork tenderloin, and lettuce using the lytic bacteriophage vB_PMG_YP1. Food Research International 242: 119862.
","pubmedId":"","doi":"10.1016/j.foodres.2026.119862"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13: e1005595.
","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Yao Y, Li Y, Jiang M, Sun Y, Zheng X, Xue Y, Han BZ. 2026. Discovery of a novel Lactiplantibacillus phage enhancing flavor compound production in high-temperature Daqu. Food Microbiology 140: 105183.
","pubmedId":"","doi":"10.1016/j.fm.2026.105183"},{"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":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[],"curatorReviews":[]},{"id":"8f23a371-2f14-47dc-9325-ef6e5649fe3c","decision":"revise","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Computer Sciences"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Chemistry"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium. Table: Phage sample locations and genome assembly results
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=6) with a clear center and a turbid edge; Reccee (isolated from an enriched sample) created plaques which were smaller (1.5-2 mm, n=5) and clear, while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia and Reccee exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology.
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208,andPZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no.SRX34536418,SRX34536435, andSRX34536424,respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
","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":"Bendele, M., Cobb, I., and Cresawn, S. Subclusters. Accessed June 7, 2026. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed
","pubmedId":"","doi":""},{"reference":"Bernsel A, Viklund H, Hennerdal A, Elofsson A. 2009. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Research 37: W465-W468.
","pubmedId":"","doi":"10.1093/nar/gkp363"},{"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":"Cobb ID, Cooper K, Bendele MG, Cresawn SG. Accessed June 7, 2026. Observable. https://observablehq.com/@cresawn-labs/cluster-report
","pubmedId":"","doi":""},{"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":"Gao K, Li Y, Wang Y, Zheng C, Zhu K, Mao X, et al., Ni. 2026. Enhancing the Antibacterial Activity of Endolysin against\n Klebsiella pneumoniae\n through Fusion Engineering Using Antimicrobial Peptide Sub5. Journal of Microbiology and Biotechnology 36: 10.4014/jmb.2601.01025.
","pubmedId":"","doi":"10.4014/jmb.2601.01025"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hallgren J, Tsirigos KD, Pedersen MD, Almagro Armenteros JJ, Marcatili P, Nielsen H, Krogh A, Winther O. 2022. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. : 10.1101/2022.04.08.487609.
","pubmedId":"","doi":"10.1101/2022.04.08.487609"},{"reference":"Hirokawa T, Boon-Chieng S, Mitaku S. 1998. SOSUI: classification and secondary structure prediction system for membrane proteins.. Bioinformatics 14: 378-379.
","pubmedId":"","doi":"10.1093/bioinformatics/14.4.378"},{"reference":"Hutinet G, Kot W, Cui L, Hillebrand R, Balamkundu S, Gnanakalai S, et al., de Crécy-Lagard. 2019. 7-Deazaguanine modifications protect phage DNA from host restriction systems. Nature Communications 10: 10.1038/s41467-019-13384-y.
","pubmedId":"","doi":"10.1038/s41467-019-13384-y"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"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":"Li P, Wang X, Liu J, Li Z, Zheng X, Zhang Y, et al., Zhang. 2026. Antibacterial efficacy of a broad lytic spectrum phage against Salmonella in different food matrices and broilers. International Journal of Antimicrobial Agents 67: 107843.
","pubmedId":"","doi":"10.1016/j.ijantimicag.2026.107843"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.0955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Moore, M.; Hicks, K.; Murphy, C.; Frost, V. J. (2026). Successful Lysogen Formation for Subcluster F1 Phage, Lucia. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. doi:10.25334/QVBC-VJ22
","pubmedId":"","doi":""},{"reference":"Park N, Cho Y, Kang Y, Kong M. 2026. Characterization of Endolysin LysG77YL from\n Bacillus licheniformis\n -Infecting Bacteriophage G77YL and Application as an Antimicrobial Agent. Journal of Microbiology and Biotechnology 36: 10.4014/jmb.2601.01064.
","pubmedId":"","doi":"10.4014/jmb.2601.01064"},{"reference":"Pardo-Freire M, Bernabéu-Gimeno M, Chan BK, Turner PE, Sánchez-Romero I, Aguilar-Pérez M, Erro-Iribarren M, Domingo-Calap P. 2025. Resolution of acute rejection in a bilateral double-lung transplanted cystic fibrosis patient following phage intervention. ASM Case Reports 1: 10.1128/asmcr.00058-24.
","pubmedId":"","doi":"10.1128/asmcr.00058-24"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"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 B, Wood JD, Smith S. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Shimamura M, Becken B, Tansmore J, Cristinziano M, Abad L, Dedrick RM, et al., Dousa. 2025. Successful treatment of macrolide-resistant\n Mycobacterium abscessus\n infection using multi-drug regimens including dual β-lactams and phage therapy: case reports in two children. ASM Case Reports 1: 10.1128/asmcr.00087-24.
","pubmedId":"","doi":"10.1128/asmcr.00087-24"},{"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":"Vikram A, McCarty K, Callahan MT, Sullivan J, Sulakvelidze A. 2025. Biocontrol of Two Escherichia coli O157:H7 Strains and a Nonpathogenic Surrogate E. coli in Wheat Grains and Wheat Milling Operations Using a Lytic Bacteriophage Cocktail. Journal of Food Protection 88: 100557.
","pubmedId":"","doi":"10.1016/j.jfp.2025.100557"},{"reference":"Wang C, Niu Y, Yu X, Zhang P, Lu Q, Li Y, et al., Zhang. 2026. Biocontrol of multidrug-resistant Proteus mirabilis on chicken breast, pork tenderloin, and lettuce using the lytic bacteriophage vB_PMG_YP1. Food Research International 242: 119862.
","pubmedId":"","doi":"10.1016/j.foodres.2026.119862"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13: e1005595.
","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Yao Y, Li Y, Jiang M, Sun Y, Zheng X, Xue Y, Han BZ. 2026. Discovery of a novel Lactiplantibacillus phage enhancing flavor compound production in high-temperature Daqu. Food Microbiology 140: 105183.
","pubmedId":"","doi":"10.1016/j.fm.2026.105183"},{"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":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[{"reviewer":{"displayName":"Sarah Ball"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"c11d8a15-a9f2-4985-83e2-132efc0749bd","decision":"accept","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Computer Sciences"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Chemistry"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=5) with a clear center and a turbid edge (Figure 1D); Reccee (isolated from an enriched sample) created plaques that were smaller (1.5-2 mm, n=5) and clear (Figure 1E), while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7) (Figure 1F). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia (Figure 1A) and Reccee (Figure 1B) exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology (Figure 1C).
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208, and PZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no. SRX34536418, SRX34536435, and SRX34536424, respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
","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":"Bendele, M., Cobb, I., and Cresawn, S. Subclusters. Accessed June 7, 2026. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed
","pubmedId":"","doi":""},{"reference":"Bernsel A, Viklund H, Hennerdal A, Elofsson A. 2009. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Research 37: W465-W468.
","pubmedId":"","doi":"10.1093/nar/gkp363"},{"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":"Cobb ID, Cooper K, Bendele MG, Cresawn SG. Accessed June 7, 2026. Observable. https://observablehq.com/@cresawn-labs/cluster-report
","pubmedId":"","doi":""},{"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":"Gao K, Li Y, Wang Y, Zheng C, Zhu K, Mao X, et al., Ni. 2026. Enhancing the Antibacterial Activity of Endolysin against\n Klebsiella pneumoniae\n through Fusion Engineering Using Antimicrobial Peptide Sub5. Journal of Microbiology and Biotechnology 36: 10.4014/jmb.2601.01025.
","pubmedId":"","doi":"10.4014/jmb.2601.01025"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hallgren J, Tsirigos KD, Pedersen MD, Almagro Armenteros JJ, Marcatili P, Nielsen H, Krogh A, Winther O. 2022. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. : 10.1101/2022.04.08.487609.
","pubmedId":"","doi":"10.1101/2022.04.08.487609"},{"reference":"Hirokawa T, Boon-Chieng S, Mitaku S. 1998. SOSUI: classification and secondary structure prediction system for membrane proteins.. Bioinformatics 14: 378-379.
","pubmedId":"","doi":"10.1093/bioinformatics/14.4.378"},{"reference":"Hutinet G, Kot W, Cui L, Hillebrand R, Balamkundu S, Gnanakalai S, et al., de Crécy-Lagard. 2019. 7-Deazaguanine modifications protect phage DNA from host restriction systems. Nature Communications 10: 10.1038/s41467-019-13384-y.
","pubmedId":"","doi":"10.1038/s41467-019-13384-y"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"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":"Li P, Wang X, Liu J, Li Z, Zheng X, Zhang Y, et al., Zhang. 2026. Antibacterial efficacy of a broad lytic spectrum phage against Salmonella in different food matrices and broilers. International Journal of Antimicrobial Agents 67: 107843.
","pubmedId":"","doi":"10.1016/j.ijantimicag.2026.107843"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence. Nucleic Acids Research 25: 955-964.
","pubmedId":"","doi":"10.1093/nar/25.5.0955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Moore, M.; Hicks, K.; Murphy, C.; Frost, V. J. (2026). Successful Lysogen Formation for Subcluster F1 Phage, Lucia. HHMI Science Education Alliance (SEA) Faculty Group, QUBES Educational Resources. doi:10.25334/QVBC-VJ22
","pubmedId":"","doi":""},{"reference":"Park N, Cho Y, Kang Y, Kong M. 2026. Characterization of Endolysin LysG77YL from\n Bacillus licheniformis\n -Infecting Bacteriophage G77YL and Application as an Antimicrobial Agent. Journal of Microbiology and Biotechnology 36: 10.4014/jmb.2601.01064.
","pubmedId":"","doi":"10.4014/jmb.2601.01064"},{"reference":"Pardo-Freire M, Bernabéu-Gimeno M, Chan BK, Turner PE, Sánchez-Romero I, Aguilar-Pérez M, Erro-Iribarren M, Domingo-Calap P. 2025. Resolution of acute rejection in a bilateral double-lung transplanted cystic fibrosis patient following phage intervention. ASM Case Reports 1: 10.1128/asmcr.00058-24.
","pubmedId":"","doi":"10.1128/asmcr.00058-24"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"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 B, Wood JD, Smith S. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbrinsgd.org/login
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Shimamura M, Becken B, Tansmore J, Cristinziano M, Abad L, Dedrick RM, et al., Dousa. 2025. Successful treatment of macrolide-resistant\n Mycobacterium abscessus\n infection using multi-drug regimens including dual β-lactams and phage therapy: case reports in two children. ASM Case Reports 1: 10.1128/asmcr.00087-24.
","pubmedId":"","doi":"10.1128/asmcr.00087-24"},{"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":"Vikram A, McCarty K, Callahan MT, Sullivan J, Sulakvelidze A. 2025. Biocontrol of Two Escherichia coli O157:H7 Strains and a Nonpathogenic Surrogate E. coli in Wheat Grains and Wheat Milling Operations Using a Lytic Bacteriophage Cocktail. Journal of Food Protection 88: 100557.
","pubmedId":"","doi":"10.1016/j.jfp.2025.100557"},{"reference":"Wang C, Niu Y, Yu X, Zhang P, Lu Q, Li Y, et al., Zhang. 2026. Biocontrol of multidrug-resistant Proteus mirabilis on chicken breast, pork tenderloin, and lettuce using the lytic bacteriophage vB_PMG_YP1. Food Research International 242: 119862.
","pubmedId":"","doi":"10.1016/j.foodres.2026.119862"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13: e1005595.
","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Yao Y, Li Y, Jiang M, Sun Y, Zheng X, Xue Y, Han BZ. 2026. Discovery of a novel Lactiplantibacillus phage enhancing flavor compound production in high-temperature Daqu. Food Microbiology 140: 105183.
","pubmedId":"","doi":"10.1016/j.fm.2026.105183"},{"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":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[],"curatorReviews":[]},{"id":"7569f62a-9994-4928-ab3d-14b88c764bac","decision":"revise","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Computer Sciences"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Chemistry"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=5) with a clear center and a turbid edge (Figure 1D); Reccee (isolated from an enriched sample) created plaques that were smaller (1.5-2 mm, n=5) and clear (Figure 1E), while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7) (Figure 1F). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia (Figure 1A) and Reccee (Figure 1B) exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology (Figure 1C).
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208, and PZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no. SRX34536418, SRX34536435, and SRX34536424, respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
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","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Yao Y, Li Y, Jiang M, Sun Y, Zheng X, Xue Y, Han BZ. 2026. Discovery of a novel Lactiplantibacillus phage enhancing flavor compound production in high-temperature Daqu. Food Microbiology 140: 105183.
","pubmedId":"","doi":"10.1016/j.fm.2026.105183"},{"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":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[],"curatorReviews":[]},{"id":"44fd907e-1dd8-4212-8c83-ffbde6dbf93f","decision":"publish","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Computer Sciences"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Chemistry"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=5) with a clear center and a turbid edge (Figure 1D); Reccee (isolated from an enriched sample) created plaques that were smaller (1.5-2 mm, n=5) and clear (Figure 1E), while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7) (Figure 1F). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia (Figure 1A) and Reccee (Figure 1B) exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology (Figure 1C).
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208, and PZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no. SRX34536418, SRX34536435, and SRX34536424, respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
","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.
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","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Yao Y, Li Y, Jiang M, Sun Y, Zheng X, Xue Y, Han BZ. 2026. Discovery of a novel Lactiplantibacillus phage enhancing flavor compound production in high-temperature Daqu. Food Microbiology 140: 105183.
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","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Complete Genome Sequences and Characteristics of Mycobacterium smegmatis Phages Lucia, Cleatunium, and Reccee
","reviews":[],"curatorReviews":[]},{"id":"afeb1bb3-df76-4dd5-ad4d-12dfe90c4a8d","decision":"publish","abstract":"Bacteriophages Lucia, Reccee, and Cleatunium were isolated from soil beneath bushes at Winthrop University using the host bacterium Mycobacterium smegmatis mc2 155. Both Lucia and Reccee exhibit siphovirus morphology, with genome sizes of 54,700 bp and 70,134 bp, respectively. Cleatunium has a myovirus morphology and a genome size of 155,948 bp. Lucia can form lysogens, whereas both Reccee and Cleatunium are predicted to replicate only via the lytic cycle. Lucia is classified in the F1 subcluster, Reccee belongs to the B5 subcluster, and Cleatunium is in the C1 subcluster.
","acknowledgements":"We thank the Howard Hughes Medical Institute SEA program for its continued support (especially Dan Russell and Rebecca Garlena at the University of Pittsburgh). We also thank Ms. Sigmon at the University of South Carolina (Columbia) for providing transmission electron microscopy (TEM) micrographs. Undergraduate students in the 2025-26 SEA-PHAGES program at Winthrop University, Rock Hill, SC, collected and annotated the phages described here.
","authors":[{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"frostv@winthrop.edu","firstName":"Victoria J","lastName":"Frost","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0003-4929-0873"},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"bagleyc3@mailbox.winthrop.edu","firstName":"Cadyn E","lastName":"Bagley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Computer Sciences"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"beasleyl3@mailbox.winthrop.edu","firstName":"Logan A","lastName":"Beasley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"leed17@mailbox.winthrop.edu","firstName":"Dylan C","lastName":"Lee","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Chemistry"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"martink31@mailbox.winthrop.edu","firstName":"Kelsey G","lastName":"Martin","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"matthewse8@mailbox.winthrop.edu","firstName":"Elizabeth R","lastName":"Matthews","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"moorem45@mailbox.winthrop.edu","firstName":"Myles A","lastName":"Moore","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"nightingalee2@mailbox.winthrop.edu","firstName":"Emmie D","lastName":"Nightingale","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"phamn4@mailbox.winthrop.edu","firstName":"Nathaniel S ","lastName":"Pham","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"toirkense2@mailbox.winthrop.edu","firstName":"Emarie M","lastName":"Toirkens","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"uribehuizachek2@mailbox.winthrop.edu","firstName":"Karen ","lastName":"Uribe-Huizache","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["Winthrop University, Rock Hill, SC, USA"],"departments":["Biology"],"credit":["supervision","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"westoverk@winthrop.edu","firstName":"Kristi M","lastName":"Westover","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0000-0002-4863-4331"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/de86fb45a9d707ca5104903dfe0bb917.csv"},"extendedData":[],"funding":"V.F. acknowledges support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM103499. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
","image":{"url":"https://portal.micropublication.org/uploads/6ecd0a1b07a9f7f0b8fdfa03bb3a61c7.png"},"imageCaption":"Transmission electron micrographs of mycobacterium phages Lucia (A), Reccee (B), and Cleatunium (C). Phage lysates were negatively stained with 1% uranyl acetate, and images were taken with a JEOL JEM-1230 TEM at 100kV acceleration voltage. Lucia (D) forms clear plaques with a turbid edge (3-5 mm, n=5). Reccee (E) forms clear plaques (1.5-2 mm, n=5), and Cleatunium (F) forms relatively large, clear plaques (4-6 mm, n=7).
Table: Phage sample locations and genome assembly results
","imageTitle":"Particle and plaque morphologies for Lucia, Reccee, and Cleatunium
","methods":"","reagents":"","patternDescription":"Our understanding of bacteriophage biology is enhanced by large-scale phage isolation and characterization efforts (Pope et al., 2015). Such efforts advance therapeutic phage applications (Shimamura et al., 2025; Pardo-Freire et al., 2025), phage use during food processing and biocontrol (Vikram et al., 2025; Wang et al., 2026; Li et al., 2026), and the discovery of valuable phage-derived products or proteins for industrial use (Park et al., 2026; Yao et al., 2026; Gao et al., 2026). Here, we report the isolation of three phages on the bacterial host Mycobacterium smegmatis mc2 155.
Lucia was discovered in the soil among bushes along the banks of Winthrop University’s lake, while Reccee and Cleatunium were obtained from damp earth in the flower beds of the Little Chapel on campus (GPS coordinates are listed in Table 1). To isolate potential mycobacteriophages, each sample was shaken for 24 hours at 24 °C in buffer (10% glycerol, 1 mM CaCl2), then centrifuged at 4,000 rpm for 10 minutes. Supernatants were filtered (0.22 µm), and standard plaque assays were used to isolate, purify, and amplify all three phages (Zorawik et al., 2024) from both direct and enriched samples. The procedure for direct (unenriched) samples combined each filtrate with M. smegmatis, suspended in molten 7H9 agar; the mixture was overlaid onto 7H9 agar plates and incubated overnight at 37 °C. For enriched samples, filtrates were mixed with M. smegmatis in 7H9 medium and incubated for 4 days at 37 °C with shaking (250 rpm). Potential mycobacteriophages were collected following high-speed centrifugation (14,000 rpm) and filtration (0.22 µm) of the supernatant. The filtrate was then spotted onto an M. smegmatis lawn (7H9 agar plate) and incubated overnight at 37 °C. Lucia (isolated from a direct sample) created medium-sized plaques (3-5 mm, n=5) with a clear center and a turbid edge (Figure 1D); Reccee (isolated from an enriched sample) created plaques that were smaller (1.5-2 mm, n=5) and clear (Figure 1E), while Cleatunium (isolated from a direct sample) also produced clear plaques that were comparatively larger (4-6 mm, n=7) (Figure 1F). Lucia's ability to produce lysogens was confirmed experimentally (Moore et al., 2026). Both Lucia (Figure 1A) and Reccee (Figure 1B) exhibited siphovirus morphology with long, flexible tails (Lucia: 193-197 nm, n=4; Reccee: 327-335 nm, n=5) under transmission electron microscopy (TEM). The micrograph of Cleatunium showed a short, contractile tail (84-87 nm, n=4) characteristic of myovirus morphology (Figure 1C).
After two rounds of purification, each phage was amplified to generate lysates for genome extraction. Genomic DNA was extracted using the Wizard DNA Cleanup kit (Promega), and libraries prepared using the NEB Ultra II FS kit before being sequenced on an Illumina NextSeq 1000 (XLEAP-P1 kit). The resulting raw reads (100 bp) were trimmed with cutadapt v4.7 (using the option: –nextseq-trim 30) (Martin, 2011) and filtered with skewer v0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Jiang et al., 2014). The reads were then assembled with Unicycler v0.5.1 and Consed v29 (Gordon and Green, 2013; Wick et al., 2017). Table 1 describes the sequencing details and characteristics of each sequenced phage genome, including the number of putative genes, tRNAs, genome ends, and cluster assignments. Clusters were assigned based on gene content similarity (GCS), with a minimum of 35% similarity to all other sequenced phages in the Actinobacteriophage database (https://phagesdb.org/) (Russell and Hatfull, 2017; Pope et al., 2017).
Bioinformatic investigations primarily used default settings for comparison and prediction tools unless otherwise indicated. Phage genome sequences were annotated with PECAAN (v20250130) (Rinehart et al., 2016), together with Glimmer v3.02 (Delcher et al., 2007), GeneMark v4.28 (Besemer and Borodovsky, 2005), Starterator v558 (http://phages.wustl.edu/starterator/), and Phamerator (Cresawn et al., 2011), using the Actino_draft database (v657). Further analyses involved BLAST (Altschul et al., 1990) searches against the Actinobacteriophage and NCBI non-redundant databases, as well as HHpred (Söding et al., 2005) searches against the PDBmmCIF70, Pfam v37, and NCBI Conserved Domains v3.2 databases. Transmembrane domains were predicted using TOPCONS v2 (Bernsel et al., 2009), DeepTMHMM v1.0.44 (integrated into PECAAN) (Hallgren et al., 2022), and SOSUI v1.11 (Hirokawa et al., 1998). Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.6 (Lowe and Eddy, 1997) were used to identify transfer RNAs.
Annotation of phages Reccee and Cleatunium showed no identifiable immunity-repressor or integrase functions, suggesting the exclusive use of the lytic pathway for replication. However, Lucia’s genome revealed putative genes involved in lysogeny, including a tyrosine integrase, Cro, and an immunity repressor, and further experimental analysis demonstrated that Lucia could form lysogens (Moore et al., 2026). In all three mycobacteriophage genomes, many of the putative gene functions relate to phage structure, including capsids, tail proteins, and portal proteins. Other predicted functions include those involved in DNA genome packaging (HNH endonuclease) and replication (DNA polymerase I and/or DnaQ-like (DNA polymerase III subunit) proteins). Lucia (cluster F1) has a highly conserved predicted gene described as the mycobacteriophage mobile element 1 (MPME 1), which has been annotated across six phage clusters (Bendele et al., 2026; Cobb et al., 2026). Cleatunium (cluster C1) possesses a typical C1 cluster genome, predicted to encode numerous tRNAs (35), a tmRNA, and a putative baseplate J protein. Reccee (cluster B5) encodes a putative gene predicted to be a dpdA-like tRNA-guanine transglycosylase, thought to protect phage DNA from restriction enzymes (Hutinet et al., 2019). This gene function has been predicted in one other cluster B5 phage (Mysterious).
Data availability
The complete genome sequences of phages Lucia, Reccee, and Cleatunium are available in GenBank (accession no. PZ531205, PZ531208, and PZ789165, respectively). The raw sequencing reads are available in the NCBI SRA under accession no. SRX34536418, SRX34536435, and SRX34536424, respectively. The Actinobacteriophage sequencing BioProject accession number is PRJNA488469.
","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":"Bendele, M., Cobb, I., and Cresawn, S. Subclusters. Accessed June 7, 2026. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed
","pubmedId":"","doi":""},{"reference":"Bernsel A, Viklund H, Hennerdal A, Elofsson A. 2009. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Research 37: W465-W468.
","pubmedId":"","doi":"10.1093/nar/gkp363"},{"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":"Cobb ID, Cooper K, Bendele MG, Cresawn SG. Accessed June 7, 2026. Observable. https://observablehq.com/@cresawn-labs/cluster-report
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