--- id: "edwards-2021-tripl3t-zeuska" title: "Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska" authors: - "Faith Cox" - "Tiffany Lujan" - "Matthew Bristerpostma" - "Rheaven Sandoval" - "Haze Murphy" - "Leah Dowell" - "Jaime Merrill" - "Julie Edwards" - "Dustin Edwards" venue: "Microbiology Resource Announcements" year: 2021 date: "2021-08-12" doi: "10.1128/mra.00558-21" url: "/research/publications/10-1128-mra-00558-21/" pdf: "/research/publications/10-1128-mra-00558-21/dustin-edwards-10-1128-mra-00558-21.pdf" pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC8359786/" openAccess: true license: "cc-by" accessions: - "genbank:MK524499" - "genbank:MK524506" - "sra:SRX4721438" - "sra:SRX4721441" citedBy: 0 citedBySource: "OpenAlex, read 2026-09-12" --- # Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska Genomes of phages Tripl3t and Zeuska, annotated by Bluff Dale and Tolar high school students; about 53,600 bp each. ## Abstract Tripl3t and Zeuska are siphoviral bacteriophages that were isolated from Mycobacterium smegmatis mc 2 155 and contain double-stranded DNA genomes 53,565 bp and 53,598 bp in length, respectively. Tripl3t and Zeuska were annotated by students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) in community engagement with Tarleton State University. ## Full text Machine-extracted from the PDF linked above. It carries the artifacts that come with reading a typeset two-column page: running heads, figure captions in the flow of the prose, and words broken across line ends. The abstract above is the registry's deposit and is the authoritative text. Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska Faith Cox,a Tiffany Lujan,a Matthew Bristerpostma,a Rheaven Sandoval,a Haze Murphy,a Leah Dowell,a Jaime Merrill,a Julie Edwards,a Dustin Edwardsa aDepartment of Biological Sciences, Tarleton State University, Stephenville, Texas, USA ABSTRACT Tripl3t and Zeuska are siphoviral bacteriophages that were isolated from Mycobacterium smegmatis mc2 155 and contain double-stranded DNA genomes 53,565 bp and 53,598 bp in length, respectively. Tripl3t and Zeuska were annotated by students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) in community engagement with Tarleton State University. Mycobacteriophages from the Howards Hughes Medical Institute Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA- PHAGES) library have been used previously to successfully treat Mycobacterium infec- tions (1, 2). As part of the SEA-PHAGES program, we report the genomes of Siphoviridae mycobacteriophages Tripl3t and Zeuska, which were isolated from soil samples from Washington, DC (38.92426N, 77.01955W), and Providence, Rhode Island (41.824047N, 71.403114W), respectively. Mycobacteriophage Tripl3t was originally isolated by Demi I. F. Lewis at Howard University (Washington, DC), and mycobacteriophage Zeuska was originally isolated by Emma Herold at Brown University (Providence, RI). The samples were incubated in 7H9 liquid medium at 37°C for 2 h before the supernatant was centri- fuged, filtered through a 0.22- mm filter, and incubated with Mycobacterium smegmatis mc2 155 at 37°C on Luria agar plates. Bacteriophages were isolated by two rounds of picking a single, well-separated plaque, followed by dilution of samples in a 10-fold dilu- tion series and plating with M. smegmatis mc2 155 (3). Tripl3t formed large bullseye pla- ques, while Zeuska formed medium plaques with cloudy borders (Fig. 1). High-titer lysates were obtained by flooding “webbed” plates, as described in the Phage Discovery Guide, and DNA was extracted using the Promega Wizard DNA clean-up system (3). High-titer lysates (with dimethyl sulfoxide at a final concentration of 6.7%) were shipped on dry ice to and from the Pittsburgh Bacteriophage Institute and stored at 280°C. The Tripl3t genomic library was prepared by Virginia Commonwealth University Nucleic Acids Research Facilities using the 454 DNA library preparation kit and was sequenced with a Roche 454 GS FLX sequencer to approximately 795-fold coverage from 104,617 total reads (average read length, 165 bp). The Zeuska genomic library was prepared using the TruSeq DNA nanokit and was sequenced with an Illumina MiSeq system at the Pittsburgh Bacteriophage Institute to approximately 3,844-fold coverage from 749,005 total reads (average read length, 150 bp) (4). Raw reads were assembled to produce sin- gle-bacteriophage contigs using Newbler v2.7, and Consed v22.0 was used to check for completeness, accuracy, and genome termini (4, 5). Tripl3t and Zeuska have linear dou- ble-stranded genomes of 53,565 bp and 53,598 bp, respectively. Both bacteriophage genomes have a G1C content of 63.7% and a 10-base 39 sticky overhang with the sequence 59-CGGATGGTAA-39. Whole-genome nucleotide alignments by BLASTn (https://blast.ncbi.nlm.nih.gov) showed Tripl3t and Zeuska to have close nucleotide identity to subcluster A1 bacterio- phages Wheeler (GenBank accession number NC_022070) and Abrogate (GenBank Citation Cox F, Lujan T, Bristerpostma M, Sandoval R, Murphy H, Dowell L, Merrill J, Edwards J, Edwards D. 2021. Complete genome sequences of Mycobacterium phages Tripl3t and Zeuska. Microbiol Resour Announc 10:e00558-21. https://doi.org/10.1128/MRA .00558-21. Editor Simon Roux, DOE Joint Genome Institute Copyright © 2021 Cox et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Dustin Edwards, dcedwards@tarleton.edu. Received 30 May 2021 Accepted 21 July 2021 Published 12 August 2021 Volume 10 Issue 32 e00558-21 mra.asm.org 1 GENOME SEQUENCES Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207. accession number KM597531) (6–8). Initial autoannotations were generated using Glimmer v3.02 (9) and GeneMark v2.5p (10), with manual revisions using DNA Master v5.23.2 (http://phagesdb.org/DNAMaster) and PECAAN (https://pecaan.kbrinsgd.org). No tRNA genes were detected by ARAGORN v1.2.38 (11) or tRNAscan-SE v2.0 (12). Putative gene functions were assigned with HHpred v3.0beta (13, 14) and BLASTp (8). All tools were run with default parameters. Putative functions were assigned to 36 of 91 predicted protein-coding genes for Tripl3t and 35 of 95 predicted protein-coding genes for Zeuska. Both bacteriophage genomes contain genes for virion assembly and structure, lysis proteins, host integration and excision proteins, DNA primase, RusA-like resolvase, RtcB-like ligase, integrase, and an immunity repressor. Data availability. GenBank and SRA accession numbers are as follows: Tripl3t, GenBank accession number MK524499 and SRA accession number SRX4721441; Zeuska, GenBank accession number MK524506 and SRA accession number SRX4721438. ACKNOWLEDGMENTS Support for this research was provided by Tarleton State University College of Science and Technology and the Howard Hughes Medical Institute SEA-PHAGES research and education program. We thank Graham Hatfull, Welkin Pope, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena, and Sally Molloy for their technical support during the sequencing and annotation of these genomes. We also thank students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) who participated in the annotation of these genomes as part of a Tarleton State University science outreach program. FIG 1 Images of plaques from bacteriophages Tripl3t (A) and Zeuska (B) incubated with Mycobacterium smegmatis mc2 155 on Luria agar plates. After 24 h of incubation at 37°C, Tripl3t formed mostly large bullseye plaques and Zeuska formed medium plaques with cloudy borders. Cox et al. Volume 10 Issue 32 e00558-21 mra.asm.org 2 Downloaded from https://journals.asm.org/journal/mra on 26 July 2026 by 156.146.253.207. REFERENCES 1. 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