{ "componentChunkName": "component---src-templates-article-page-js", "path": "/journals/biology/micropub-biology-002379", "result": {"data":{"article":{"manuscript":{"id":"0e3dc7cf-1ef2-4652-8ced-a04cb4d0b9d6","submissionTypes":["new finding"],"citations":[],"doi":"10.17912/micropub.biology.002379","dbReferenceId":null,"pmcId":null,"pmId":null,"proteopedia":null,"reviewPanel":null,"species":["bacteriophage"],"integrations":[],"corrections":null,"history":{"received":"2026-08-26T15:48:12.146Z","revisionReceived":"2026-10-05T07:22:49.862Z","accepted":"2026-10-20T18:16:19.683Z","published":"2026-10-29T14:33:43.413Z","indexed":"2026-11-12T14:33:43.413Z"},"versions":[{"id":"444e2efe-be7b-4ca9-b1ed-20c3f8ab7178","decision":"revise","abstract":"

MajinBuu and Coldpizza are bacteriophages isolated on Gordonia rubripertincta NRRL B-16540. MajinBuu has a 61,525 bp genome with a 3’ overhang and 92 predicted genes, while Coldpizza’s 61,651 bp circularly permuted genome encodes 79 predicted genes. Neither genome is predicted to contain genes encoding tRNAs or tmRNAs.

","acknowledgements":"

This research was supported by the Biological Sciences Department of the College of Southern Nevada. We thank the SEA-PHAGES program and the University of Pittsburgh for the opportunity to study bacteriophages and for sequencing the phage genomes for annotation. We thank Navya Reddy for discovering Coldpizza.

","authors":[{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"sarah.beattye@gmail.com","firstName":"Sarah E","lastName":"Beatty","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"maritza06cervantes@gmail.com","firstName":"Maritza","lastName":"Cervantes","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"fanamite95@gmail.com","firstName":"Hope M","lastName":"Dunn","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"lissettegarcia0430@gmail.com","firstName":"Lissette E","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation"],"email":"gerlynfortuna@gmail.com","firstName":"Gerlyn M","lastName":"Fortuna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"esmondcfung@gmail.com","firstName":"Esmond C","lastName":"Fung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"mptf8780@gmail.com","firstName":"Maddisson S","lastName":"Harmon Petroff","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"v.c.5011x@gmail.com","firstName":"Itzayana B","lastName":"Parker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"kendra.kimberley@csn.edu","firstName":"Kendra W","lastName":"Kimberley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"chelsey.mckenna@csn.edu","firstName":"Chelsey C","lastName":"McKenna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"james.theoret@csn.edu","firstName":"James R","lastName":"Theoret","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"erin.windsor@csn.edu","firstName":"Erin J","lastName":"Windsor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"earl.yoon@csn.edu","firstName":"Earl J ","lastName":"Yoon","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0002-7302-8819"}],"awards":[],"conflictsOfInterest":"

The authors declare that there are no conflicts of interest present.

","dataTable":{"url":"https://portal.micropublication.org/uploads/9ba5f15ae47de558cb51672cf4c258c7.csv"},"extendedData":[],"funding":"

Funding for this work was provided by the Biological Sciences Department of the College of Southern Nevada.

","image":{"url":null},"imageCaption":"

Table 1. Actinobacteriophage genome and sequencing information.

","imageTitle":"

Genomic Characteristics of Bacteriophages MajinBuu and Coldpizza

","methods":"

","reagents":"

","patternDescription":"

Bacteriophages are diverse and abundant in terrestrial systems, and it is estimated that there are 109 phages per gram of soil (Hatfull, 2015). MajinBuu and Coldpizza are two bacteriophages that infect the host bacterium, Gordonia rubripertincta NRRL B-16540. They were isolated from soil and characterized through the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Russell and Hatfull, 2017). Gordonia are bacteria commonly found in soil and associated with wastewater in treatment plants. They are also opportunistic pathogens that can cause catheter infections and other infections in immunocompromised patients (Pollenz et al., 2022).

MajinBuu and Coldpizza were isolated from soil samples collected in a park in Las Vegas (GPS coordinates: 36.28765 N, 115.31643 W) using a direct isolation method and from a plant bed in Pittsburgh (GPS coordinates: 40.440107 N, 79.958725 W) using an enriched isolation method, respectively (Zorawik et al., 2024). Briefly, the MajinBuu soil sample was incubated in nutrient broth with Amphotericin B and CaCl2, and the Coldpizza soil sample was incubated in PYCa (peptone-yeast-calcium) broth. Both suspensions were incubated at 30°C with shaking at 250rpm for one hour. The suspensions were spun in a centrifuge at 2,000 x g for 10 minutes, and the supernatants were filtered through 0.22μm filters. For direct isolation, the supernatant was used directly for plaque assays by plating in top agar with Gordonia rubripertincta NRRL B-16540. For enriched isolation, 10mL of supernatant was inoculated with 500μL of G. rubripertincta and incubated at 30°C for three days on a rocker at 220rpm. The resulting culture was filtered through a 0.22μm filter prior to the plaque assay. After completing three rounds of picking isolated plaques and plating to purify the phages, and preparing liquid lysates of these phages, DNA was extracted from the lysates using the Norgen phage DNA isolation kit, which included five rounds of freeze/thaw (4-minute freeze in a dry-ice methanol bath and a 1-minute thaw in 60°C water).

Sequencing libraries were created from extracted genomic DNA using New England Biolabs Ultra II Library kit, before sequencing at the Pittsburgh Bacteriophage Institute using an Illumina NextSeq 1000 instrument (X-LEAP P1 kit). The resulting100-base single-end reads were trimmed by cutadapt 4.7 (using option:  –nextseq-trim 30) and skewer 0.2.2 (using options: -q 20 -Q 30 -n -l 50) and then assembled using Unicycler version 0.5.1 (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Assemblies were visualized using Consed v29 and checked for accuracy and completeness by looking for gaps and low consensus areas  (Gordon et al., 1998). Bacteriophage genomic termini were determined via analysis of read start pileups and coverage as previously described (Russell, 2018). Sequencing and assembly results are presented in Table 1.

Annotations were completed using the following programs: DNA Master v2.53.6 (http://cobamide2.bio.pitt.edu/computer.htm), Starterator v1.2 (https://github.com/SEA-PHAGES/starterator), Phamerator (https://phamerator.org/) (Cresawn et al., 2011), PhagesDB BLAST (database: actinobacteriophage database; https://phagesdb.org) (Russell and Hatfull, 2017), NCBI BLAST (Databases: blastn standard core nucleotide and blastp standard non-redundant) (Altschul et al., 1990), PECAAN (Rinehart et al., 2016), GeneMark v2.5p (Besemer and Borodovsky, 2005), Glimmer v3.02 (Delcher et al., 2007), Aragorn (v1.1 and v1.2.38) (Laslett and Canback, 2004), HHPRED (Databases: PDB, NCBI-CD, PFam-A, and SCOPe) (Gabler et al., 2020), tRNAscan-SE v2.0 (Lowe and Chan, 2016), DeepTMHMM v1.0.24 (Hallgren et al., 2022), all using the default settings.

MajinBuu has 92 predicted genes that are transcribed unidirectionally, and 26 of these genes were assigned putative functions. Coldpizza has 79 genes, and 31 were assigned putative functions. MajinBuu and Coldpizza were assigned to clusters DJ and DR, respectively, based on gene content similarity of at least 35% to the phages in the Actinobacteriophage database, phagesdb (https://phageDB.org) (Pope et al., 2017; Russell and Hatfull, 2017). As with other cluster DJ phages, genes associated with lysis in MajinBuu, including lysA, lysB, and holin, are flanked by a conserved set of genes encoding proteins of unknown function that contain transmembrane domains. The proximity and synteny of these transmembrane-containing proteins to lysis genes suggests involvement in lysis and may define a lysis cassette (Bendele et al., 2025). Unlike all cluster DR phages, where both lysA and lysB genes could be identified, no lysB could be identified for ColdPizza. Where Lysin A is typically essential for phage replication, Lysin B is not (Payne et al., 2009). Both genomes do not contain any predicted tRNA or tmRNA genes, which is typical for both clusters. Similarly, neither phage is predicted to be lysogenic due to the absence of identifiable immunity repressor or integrase genes.

Data Availability. MajinBuu GenBank and SRA accession numbers are PV915850 and SRR34858428, respectively. Coldpizza GenBank and SRA accession numbers are PV915803 and SRR34858419, respectively.

","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, 2025. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed

","pubmedId":"","doi":""},{"reference":"

Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.

","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"

Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.

","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"

Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.

","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"

Gabler F, Nam SZ, Till S, Mirdita M, Steinegger M, Söding J, Lupas AN, Alva V. 2020. Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Current Protocols in Bioinformatics 72: 10.1002/cpbi.108.

","pubmedId":"","doi":"10.1002/cpbi.108"},{"reference":"

Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.

","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"

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":"

Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.

","pubmedId":"","doi":"10.1128/jvi.01340-15"},{"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, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32(1): 11-6.

","pubmedId":"14704338","doi":""},{"reference":"

Lowe TM, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.

","pubmedId":"","doi":"10.1093/nar/gkw413"},{"reference":"

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":"

Rinehart, CA, Gaffney, B, Wood, JD, & Smith, J. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbringsd.org/login

","pubmedId":"","doi":""},{"reference":"

Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33(5): 784-786.

","pubmedId":"28365761","doi":""},{"reference":"

Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"

Payne K, Sun Q, Sacchettini J, Hatfull GF. 2009. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase. Molecular Microbiology 73: 367-381.

","pubmedId":"","doi":"10.1111/j.1365-2958.2009.06775.x"},{"reference":"

Pollenz RS, Bland J, Pope WH. 2022. Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta. PLOS ONE 17: e0276603.

","pubmedId":"","doi":"10.1371/journal.pone.0276603"},{"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":"

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":"

Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.

","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"

Genome Sequences of Gordonia rubripertincta Bacteriophages MajinBuu and Coldpizza

","reviews":[{"reviewer":{"displayName":"Victoria Frost"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"fa58b4d6-388b-4468-8ce2-01729d93bcd1","decision":"accept","abstract":"

MajinBuu and Coldpizza are bacteriophages isolated on Gordonia rubripertincta NRRL B-16540. MajinBuu has a 61,525 bp genome with a 3’ overhang and 92 predicted genes, while Coldpizza’s 61,651 bp circularly permuted genome encodes 79 predicted genes. Neither genome is predicted to contain genes encoding tRNAs or tmRNAs.

","acknowledgements":"

This research was supported by the Biological Sciences Department of the College of Southern Nevada. We thank the SEA-PHAGES program and the University of Pittsburgh for the opportunity to study bacteriophages and for sequencing the phage genomes for annotation. We thank Navya Reddy for discovering Coldpizza.

","authors":[{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"sarah.beattye@gmail.com","firstName":"Sarah E","lastName":"Beatty","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"maritza06cervantes@gmail.com","firstName":"Maritza","lastName":"Cervantes","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"fanamite95@gmail.com","firstName":"Hope M","lastName":"Dunn","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"lissettegarcia0430@gmail.com","firstName":"Lissette E","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation"],"email":"gerlynfortuna@gmail.com","firstName":"Gerlyn M","lastName":"Fortuna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"esmondcfung@gmail.com","firstName":"Esmond C","lastName":"Fung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"mptf8780@gmail.com","firstName":"Maddisson S","lastName":"Harmon Petroff","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"v.c.5011x@gmail.com","firstName":"Itzayana B","lastName":"Parker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"kendra.kimberley@csn.edu","firstName":"Kendra W","lastName":"Kimberley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"chelsey.mckenna@csn.edu","firstName":"Chelsey C","lastName":"McKenna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"james.theoret@csn.edu","firstName":"James R","lastName":"Theoret","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"erin.windsor@csn.edu","firstName":"Erin J","lastName":"Windsor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"earl.yoon@csn.edu","firstName":"Earl J ","lastName":"Yoon","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0002-7302-8819"}],"awards":[],"conflictsOfInterest":"

The authors declare that there are no conflicts of interest present.

","dataTable":{"url":"https://portal.micropublication.org/uploads/9ba5f15ae47de558cb51672cf4c258c7.csv"},"extendedData":[],"funding":"

Funding for this work was provided by the Biological Sciences Department of the College of Southern Nevada.

","image":{"url":null},"imageCaption":"

Information about the discovery, genome characteristics, and sequencing for MajinBuu and Coldpizza.

","imageTitle":"

Genomic Characteristics of Bacteriophages MajinBuu and Coldpizza

","methods":"

","reagents":"

","patternDescription":"

Bacteriophages are diverse and abundant in terrestrial systems, and it is estimated that there are 109 phages per gram of soil (Hatfull, 2015). MajinBuu and Coldpizza are two bacteriophages that infect the host bacterium, Gordonia rubripertincta NRRL B-16540. They were isolated from soil and characterized through the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Russell and Hatfull, 2017). Gordonia are bacteria commonly found in soil and associated with wastewater in treatment plants. They are also opportunistic pathogens that can cause catheter infections and other infections in immunocompromised patients (Pollenz et al., 2022).

MajinBuu and Coldpizza were isolated from soil samples collected in a park in Las Vegas (GPS coordinates: 36.28765 N, 115.31643 W) using a direct isolation method and from a plant bed in Pittsburgh (GPS coordinates: 40.440107 N, 79.958725 W) using an enriched isolation method, respectively (Zorawik et al., 2024). Briefly, the MajinBuu soil sample was incubated in nutrient broth with 2.5 μg/mL Amphotericin B and 4.5 mM CaCl2, and the Coldpizza soil sample was incubated in PYCa (peptone-yeast-calcium) broth. Both suspensions were incubated at 30°C with shaking at 250 rpm for one hour. The suspensions were spun in a centrifuge at 2,000 x g for 10 minutes, and the supernatants were filtered through 0.22 μm filters. For direct isolation, the supernatant was used directly for plaque assays by plating in top agar with Gordonia rubripertincta NRRL B-16540. For enriched isolation, 10 mL of supernatant was inoculated with 500 μL of G. rubripertincta and incubated at 30°C for three days on a rocker at 220 rpm. The resulting culture was filtered through a 0.22 μm filter prior to the plaque assay. After completing three rounds of picking isolated plaques and plating to purify the phages, and preparing liquid lysates of these phages, DNA was extracted from the lysates using the Norgen phage DNA isolation kit, which included five rounds of freeze/thaw (4-minute freeze in a dry-ice methanol bath and a 1-minute thaw in 60°C water). MajinBuu forms clear round plaques with defined borders that have an average diameter of 1.06 mm (plaque diameter based on measurements of five plaques from a single plate). Coldpizza forms smaller round clear plaques with defined borders that have an average diameter of 0.65 mm (plaque diameter based on measurements of five plaques from a single plate). Plaque measurement analysis for both phages was performed using ImageJ (Schneider et al., 2012).

Sequencing libraries were created from extracted genomic DNA using New England Biolabs Ultra II Library kit, before sequencing at the Pittsburgh Bacteriophage Institute using an Illumina NextSeq 1000 instrument (X-LEAP P1 kit). The resulting 100-base single-end reads were trimmed by cutadapt 4.7 (using option:  –nextseq-trim 30) and skewer 0.2.2 (using options: -q 20 -Q 30 -n -l 50) and then assembled using Unicycler version 0.5.1 (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Assemblies were visualized using Consed v29 and checked for accuracy and completeness by looking for gaps and low consensus areas  (Gordon et al., 1998). Bacteriophage genomic termini were determined via analysis of read start pileups and coverage as previously described (Russell, 2018). Sequencing and assembly results are presented in Table 1.

Annotations were completed using the following programs: DNA Master v2.53.6 (http://cobamide2.bio.pitt.edu/computer.htm), Starterator v1.2 (https://github.com/SEA-PHAGES/starterator), Phamerator v2.5 (Actino_Draft database, https://phamerator.org/) (Cresawn et al., 2011), PhagesDB BLAST (database: actinobacteriophage database; https://phagesdb.org) (Russell and Hatfull, 2017), NCBI BLAST (Databases: blastn standard core nucleotide and blastp standard non-redundant) (Altschul et al., 1990), PECAAN (Rinehart et al., 2016), GeneMark v2.5p (Besemer and Borodovsky, 2005), Glimmer v3.02 (Delcher et al., 2007), Aragorn (v1.1 and v1.2.38) (Laslett and Canback, 2004), HHPRED (Databases: PDB, NCBI-CD, PFam-A, and SCOPe) (Gabler et al., 2020), tRNAscan-SE v2.0 (Lowe and Chan, 2016), DeepTMHMM v1.0.24 (Hallgren et al., 2022), all using the default settings.

MajinBuu has 92 predicted genes that are transcribed unidirectionally, and 26 of these genes were assigned putative functions. Coldpizza has 79 genes, and 31 were assigned putative functions. MajinBuu and Coldpizza were assigned to clusters DJ and DR, respectively, based on gene content similarity of at least 35% to the phages in the Actinobacteriophage database, phagesdb (https://phageDB.org) (Pope et al., 2017; Russell and Hatfull, 2017). As with other cluster DJ phages, genes associated with lysis in MajinBuu, including lysA, lysB, and those encoding holin, are flanked by a conserved set of genes encoding proteins of unknown function that contain transmembrane domains. The proximity and synteny of these transmembrane-containing proteins to lysis genes suggests involvement in lysis and may define a lysis cassette (Bendele et al., 2025). Unlike all cluster DR phages, where both lysA and lysB genes could be identified, no lysB could be identified for Coldpizza. Where Lysin A is typically essential for phage replication, Lysin B is not (Payne et al., 2009). Both genomes do not contain any predicted tRNA or tmRNA genes, which is typical for both clusters. Similarly, neither phage is predicted to be lysogenic due to the absence of identifiable immunity repressor or integrase genes.

Data Availability. MajinBuu GenBank and SRA accession numbers are PV915850 and SRR34858428, respectively. Coldpizza GenBank and SRA accession numbers are PV915803 and SRR34858419, respectively.

","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, 2025. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed

","pubmedId":"","doi":""},{"reference":"

Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.

","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"

Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 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":"

Gabler F, Nam SZ, Till S, Mirdita M, Steinegger M, Söding J, Lupas AN, Alva V. 2020. Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Current Protocols in Bioinformatics 72: e108.

","pubmedId":"","doi":"10.1002/cpbi.108"},{"reference":"

Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.

","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"

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. bioRxiv 2022.04.08.487609.

","pubmedId":"","doi":"10.1101/2022.04.08.487609"},{"reference":"

Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.

","pubmedId":"","doi":"10.1128/jvi.01340-15"},{"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: 182.

","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"

Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32(1): 11-6.

","pubmedId":"14704338","doi":""},{"reference":"

Lowe TM, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.

","pubmedId":"","doi":"10.1093/nar/gkw413"},{"reference":"

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":"

Rinehart, CA, Gaffney, B, Wood, JD, & Smith, J. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbringsd.org/login

","pubmedId":"","doi":""},{"reference":"

Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33(5): 784-786.

","pubmedId":"28365761","doi":""},{"reference":"

Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"

Payne K, Sun Q, Sacchettini J, Hatfull GF. 2009. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase. Molecular Microbiology 73: 367-381.

","pubmedId":"","doi":"10.1111/j.1365-2958.2009.06775.x"},{"reference":"

Pollenz RS, Bland J, Pope WH. 2022. Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta. PLOS ONE 17: e0276603.

","pubmedId":"","doi":"10.1371/journal.pone.0276603"},{"reference":"

Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: e01069-17.

","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"

Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671-675.

","pubmedId":"","doi":"10.1038/nmeth.2089"},{"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":"

Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.

","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"

Genome Sequences of Gordonia rubripertincta Bacteriophages MajinBuu and Coldpizza

","reviews":[],"curatorReviews":[]},{"id":"7989db47-d34d-49b6-bb7d-9895ea351837","decision":"revise","abstract":"

MajinBuu and Coldpizza are bacteriophages isolated on Gordonia rubripertincta NRRL B-16540. MajinBuu has a 61,525 bp genome with a 3' overhang and 92 predicted genes, while Coldpizza's 61,651 bp circularly permuted genome encodes 79 predicted genes. Neither genome is predicted to contain genes encoding tRNAs or tmRNAs.

","acknowledgements":"

This research was supported by the Biological Sciences Department of the College of Southern Nevada. We thank the SEA-PHAGES program and the University of Pittsburgh for the opportunity to study bacteriophages and for sequencing the phage genomes for annotation. We thank Navya Reddy for discovering Coldpizza.

","authors":[{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"sarah.beattye@gmail.com","firstName":"Sarah E","lastName":"Beatty","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"maritza06cervantes@gmail.com","firstName":"Maritza","lastName":"Cervantes","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"fanamite95@gmail.com","firstName":"Hope M","lastName":"Dunn","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"lissettegarcia0430@gmail.com","firstName":"Lissette E","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation"],"email":"gerlynfortuna@gmail.com","firstName":"Gerlyn M","lastName":"Fortuna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"esmondcfung@gmail.com","firstName":"Esmond C","lastName":"Fung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"mptf8780@gmail.com","firstName":"Maddisson S","lastName":"Harmon Petroff","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"v.c.5011x@gmail.com","firstName":"Itzayana B","lastName":"Parker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["investigation"],"email":"kendra.kimberley@csn.edu","firstName":"Kendra W","lastName":"Kimberley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"chelsey.mckenna@csn.edu","firstName":"Chelsey C","lastName":"McKenna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation"],"email":"james.theoret@csn.edu","firstName":"James R","lastName":"Theoret","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"erin.windsor@csn.edu","firstName":"Erin J","lastName":"Windsor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada"],"departments":["Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"earl.yoon@csn.edu","firstName":"Earl J ","lastName":"Yoon","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0002-7302-8819"}],"awards":[],"conflictsOfInterest":"

The authors declare that there are no conflicts of interest present.

","dataTable":{"url":"https://portal.micropublication.org/uploads/9ba5f15ae47de558cb51672cf4c258c7.csv"},"extendedData":[],"funding":"

Funding for this work was provided by the Biological Sciences Department of the College of Southern Nevada.

","image":{"url":null},"imageCaption":"

Information about the discovery, genome characteristics, and sequencing for MajinBuu and Coldpizza.

","imageTitle":"

Genomic Characteristics of Bacteriophages MajinBuu and Coldpizza

","methods":"

","reagents":"

","patternDescription":"

Bacteriophages are diverse and abundant in terrestrial systems, and it is estimated that there are 109 phages per gram of soil (Hatfull, 2015). MajinBuu and Coldpizza are two bacteriophages that infect the host bacterium, Gordonia rubripertincta NRRL B-16540. They were isolated from soil and characterized through the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Russell and Hatfull, 2017). Gordonia are bacteria commonly found in soil and associated with wastewater in treatment plants. They are also opportunistic pathogens that can cause catheter infections and other infections in immunocompromised patients (Pollenz et al., 2022).

MajinBuu and Coldpizza were isolated from soil samples collected in a park in Las Vegas (GPS coordinates: 36.28765 N, 115.31643 W) using a direct isolation method and from a plant bed in Pittsburgh (GPS coordinates: 40.440107 N, 79.958725 W) using an enriched isolation method, respectively (Zorawik et al., 2024). Briefly, the MajinBuu soil sample was incubated in nutrient broth with 2.5 μg/mL Amphotericin B and 4.5 mM CaCl2, and the Coldpizza soil sample was incubated in PYCa (peptone-yeast-calcium) broth. Both suspensions were incubated at 30°C with shaking at 250 rpm for one hour. The suspensions were spun in a centrifuge at 2,000 x g for 10 minutes, and the supernatants were filtered through 0.22 μm filters. For direct isolation, the supernatant was used directly for plaque assays by plating in top agar with Gordonia rubripertincta NRRL B-16540. For enriched isolation, 10 mL of supernatant was inoculated with 500 μL of G. rubripertincta and incubated at 30°C for three days on a rocker at 220 rpm. The resulting culture was filtered through a 0.22 μm filter prior to the plaque assay. After completing three rounds of picking isolated plaques and plating to purify the phages, and preparing liquid lysates of these phages, DNA was extracted from the lysates using the Norgen phage DNA isolation kit, which included five rounds of freeze/thaw (4-minute freeze in a dry-ice methanol bath and a 1-minute thaw in 60°C water). MajinBuu forms clear round plaques with defined borders that have an average diameter of 1.06 mm (plaque diameter based on measurements of five plaques from a single plate). Coldpizza forms smaller round clear plaques with defined borders that have an average diameter of 0.65 mm (plaque diameter based on measurements of five plaques from a single plate). Plaque measurement analysis for both phages was performed using ImageJ (Schneider et al., 2012).

Sequencing libraries were created from extracted genomic DNA using New England Biolabs Ultra II Library kit, before sequencing at the Pittsburgh Bacteriophage Institute using an Illumina NextSeq 1000 instrument (X-LEAP P1 kit). The resulting 100-base single-end reads were trimmed by cutadapt 4.7 (using option:  –nextseq-trim 30) and skewer 0.2.2 (using options: -q 20 -Q 30 -n -l 50) and then assembled using Unicycler version 0.5.1 (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Assemblies were visualized using Consed v29 and checked for accuracy and completeness by looking for gaps and low consensus areas  (Gordon et al., 1998). Bacteriophage genomic termini were determined via analysis of read start pileups and coverage as previously described (Russell, 2018). Sequencing and assembly results are presented in Table 1.

Annotations were completed using the following programs: DNA Master v2.53.6 (http://cobamide2.bio.pitt.edu/computer.htm), Starterator v1.2 (https://github.com/SEA-PHAGES/starterator), Phamerator v2.5 (Actino_Draft database, https://phamerator.org/) (Cresawn et al., 2011), PhagesDB BLAST (database: actinobacteriophage database; https://phagesdb.org) (Russell and Hatfull, 2017), NCBI BLAST (Databases: blastn standard core nucleotide and blastp standard non-redundant) (Altschul et al., 1990), PECAAN (Rinehart et al., 2016), GeneMark v2.5p (Besemer and Borodovsky, 2005), Glimmer v3.02 (Delcher et al., 2007), Aragorn (v1.1 and v1.2.38) (Laslett and Canback, 2004), HHPRED (Databases: PDB, NCBI-CD, PFam-A, and SCOPe) (Gabler et al., 2020), tRNAscan-SE v2.0 (Lowe and Chan, 2016), DeepTMHMM v1.0.24 (Hallgren et al., 2022), all using the default settings.

MajinBuu has 92 predicted genes that are transcribed unidirectionally, and 26 of these genes were assigned putative functions. Coldpizza has 79 genes, and 31 were assigned putative functions. MajinBuu and Coldpizza were assigned to clusters DJ and DR, respectively, based on gene content similarity of at least 35% to the phages in the Actinobacteriophage database, phagesdb (https://phageDB.org) (Pope et al., 2017; Russell and Hatfull, 2017). As with other cluster DJ phages, genes associated with lysis in MajinBuu, including lysA, lysB, and those encoding holin, are flanked by a conserved set of genes encoding proteins of unknown function that contain transmembrane domains. The proximity and synteny of these transmembrane-containing proteins to lysis genes suggests involvement in lysis and may define a lysis cassette (Bendele et al., 2025). Unlike all cluster DR phages, where both lysA and lysB genes could be identified, no lysB could be identified for Coldpizza. Where Lysin A is typically essential for phage replication, Lysin B is not (Payne et al., 2009). Both genomes do not contain any predicted tRNA or tmRNA genes, which is typical for both clusters. Similarly, neither phage is predicted to be lysogenic due to the absence of identifiable immunity repressor or integrase genes.

Data Availability. MajinBuu GenBank and SRA accession numbers are PV915850 and SRR34858428, respectively. Coldpizza GenBank and SRA accession numbers are PV915803 and SRR34858419, respectively.

","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, 2025. Observable. https://observablehq.com/d/5e5bc78c9b3ae2ed

","pubmedId":"","doi":""},{"reference":"

Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.

","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"

Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 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":"

Gabler F, Nam SZ, Till S, Mirdita M, Steinegger M, Söding J, Lupas AN, Alva V. 2020. Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Current Protocols in Bioinformatics 72: e108.

","pubmedId":"","doi":"10.1002/cpbi.108"},{"reference":"

Gordon D, Abajian C, Green P. 1998. Consed: A Graphical Tool for Sequence Finishing. Genome Research 8: 195-202.

","pubmedId":"","doi":"10.1101/gr.8.3.195"},{"reference":"

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. bioRxiv 2022.04.08.487609.

","pubmedId":"","doi":"10.1101/2022.04.08.487609"},{"reference":"

Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.

","pubmedId":"","doi":"10.1128/jvi.01340-15"},{"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: 182.

","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"

Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32(1): 11-6.

","pubmedId":"14704338","doi":""},{"reference":"

Lowe TM, Chan PP. 2016. tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Research 44: W54-W57.

","pubmedId":"","doi":"10.1093/nar/gkw413"},{"reference":"

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":"

Rinehart, CA, Gaffney, B, Wood, JD, & Smith, J. 2016. PECAAN, a Phage Evidence Collection And Annotation Network. https://discover.kbringsd.org/login

","pubmedId":"","doi":""},{"reference":"

Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33(5): 784-786.

","pubmedId":"28365761","doi":""},{"reference":"

Russell DA. 2017. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods in Molecular Biology,Bacteriophages : 109-125.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"

Payne K, Sun Q, Sacchettini J, Hatfull GF. 2009. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase. Molecular Microbiology 73: 367-381.

","pubmedId":"","doi":"10.1111/j.1365-2958.2009.06775.x"},{"reference":"

Pollenz RS, Bland J, Pope WH. 2022. Bioinformatic characterization of endolysins and holin-like membrane proteins in the lysis cassette of phages that infect Gordonia rubripertincta. PLOS ONE 17: e0276603.

","pubmedId":"","doi":"10.1371/journal.pone.0276603"},{"reference":"

Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: e01069-17.

","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"

Schneider CA, Rasband WS, Eliceiri KW. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9: 671-675.

","pubmedId":"","doi":"10.1038/nmeth.2089"},{"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":"

Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.

","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"

Genome Sequences of Gordonia rubripertincta Bacteriophages MajinBuu and Coldpizza

","reviews":[],"curatorReviews":[]},{"id":"f4a3c8b2-fe19-4d45-9645-3fc3c4899ea4","decision":"publish","abstract":"

MajinBuu and Coldpizza are bacteriophages isolated on Gordonia rubripertincta NRRL B-16540. MajinBuu has a 61,525 bp genome with a 3' overhang and 92 predicted genes, while Coldpizza's 61,651 bp circularly permuted genome encodes 79 predicted genes. Neither genome is predicted to contain genes encoding tRNAs or tmRNAs.

","acknowledgements":"

This research was supported by the Biological Sciences Department of the College of Southern Nevada. We thank the SEA-PHAGES program and the University of Pittsburgh for the opportunity to study bacteriophages and for sequencing the phage genomes for annotation. We thank Navya Reddy for discovering Coldpizza.

","authors":[{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"sarah.beattye@gmail.com","firstName":"Sarah E","lastName":"Beatty","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"maritza06cervantes@gmail.com","firstName":"Maritza","lastName":"Cervantes","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"fanamite95@gmail.com","firstName":"Hope M","lastName":"Dunn","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"lissettegarcia0430@gmail.com","firstName":"Lissette E","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation"],"email":"gerlynfortuna@gmail.com","firstName":"Gerlyn M","lastName":"Fortuna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"esmondcfung@gmail.com","firstName":"Esmond C","lastName":"Fung","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"mptf8780@gmail.com","firstName":"Maddisson S","lastName":"Harmon Petroff","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft"],"email":"v.c.5011x@gmail.com","firstName":"Itzayana B","lastName":"Parker","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["investigation"],"email":"kendra.kimberley@csn.edu","firstName":"Kendra W","lastName":"Kimberley","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation"],"email":"chelsey.mckenna@csn.edu","firstName":"Chelsey C","lastName":"McKenna","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation"],"email":"james.theoret@csn.edu","firstName":"James R","lastName":"Theoret","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"erin.windsor@csn.edu","firstName":"Erin J","lastName":"Windsor","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Southern Nevada, Las Vegas, NV, United States"],"departments":["Department of Biological Sciences"],"credit":["validation","investigation","writing_originalDraft","writing_reviewEditing","supervision"],"email":"earl.yoon@csn.edu","firstName":"Earl J ","lastName":"Yoon","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0000-0002-7302-8819"}],"awards":[],"conflictsOfInterest":"

The authors declare that there are no conflicts of interest present.

","dataTable":{"url":"https://portal.micropublication.org/uploads/9ba5f15ae47de558cb51672cf4c258c7.csv"},"extendedData":[],"funding":"

Funding for this work was provided by the Biological Sciences Department of the College of Southern Nevada.

","image":{"url":null},"imageCaption":"

Information about the discovery, genome characteristics, and sequencing for MajinBuu and Coldpizza.

","imageTitle":"

Genomic Characteristics of Bacteriophages MajinBuu and Coldpizza

","methods":"

","reagents":"

","patternDescription":"

Bacteriophages are diverse and abundant in terrestrial systems, and it is estimated that there are 109 phages per gram of soil (Hatfull, 2015). MajinBuu and Coldpizza are two bacteriophages that infect the host bacterium, Gordonia rubripertincta NRRL B-16540. They were isolated from soil and characterized through the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program (Russell and Hatfull, 2017). Gordonia are bacteria commonly found in soil and associated with wastewater in treatment plants. They are also opportunistic pathogens that can cause catheter infections and other infections in immunocompromised patients (Pollenz et al., 2022).

MajinBuu and Coldpizza were isolated from soil samples collected in a park in Las Vegas (GPS coordinates: 36.28765 N, 115.31643 W) using a direct isolation method and from a plant bed in Pittsburgh (GPS coordinates: 40.440107 N, 79.958725 W) using an enriched isolation method, respectively (Zorawik et al., 2024). Briefly, the MajinBuu soil sample was incubated in nutrient broth with 2.5 μg/mL Amphotericin B and 4.5 mM CaCl2, and the Coldpizza soil sample was incubated in PYCa (peptone-yeast-calcium) broth. Both suspensions were incubated at 30°C with shaking at 250 rpm for one hour. The suspensions were spun in a centrifuge at 2,000 x g for 10 minutes, and the supernatants were filtered through 0.22 μm filters. For direct isolation, the supernatant was used directly for plaque assays by plating in top agar with Gordonia rubripertincta NRRL B-16540. For enriched isolation, 10 mL of supernatant was inoculated with 500 μL of G. rubripertincta and incubated at 30°C for three days on a rocker at 220 rpm. The resulting culture was filtered through a 0.22 μm filter prior to the plaque assay. After completing three rounds of picking isolated plaques and plating to purify the phages, and preparing liquid lysates of these phages, DNA was extracted from the lysates using the Norgen phage DNA isolation kit, which included five rounds of freeze/thaw (4-minute freeze in a dry-ice methanol bath and a 1-minute thaw in 60°C water). MajinBuu forms clear round plaques with defined borders that have an average diameter of 1.06 mm (plaque diameter based on measurements of five plaques from a single plate). Coldpizza forms smaller round clear plaques with defined borders that have an average diameter of 0.65 mm (plaque diameter based on measurements of five plaques from a single plate). Plaque measurement analysis for both phages was performed using ImageJ (Schneider et al., 2012).

Sequencing libraries were created from extracted genomic DNA using New England Biolabs Ultra II Library kit, before sequencing at the Pittsburgh Bacteriophage Institute using an Illumina NextSeq 1000 instrument (X-LEAP P1 kit). The resulting 100-base single-end reads were trimmed by cutadapt 4.7 (using option:  –nextseq-trim 30) and skewer 0.2.2 (using options: -q 20 -Q 30 -n -l 50) and then assembled using Unicycler version 0.5.1 (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Assemblies were visualized using Consed v29 and checked for accuracy and completeness by looking for gaps and low consensus areas  (Gordon et al., 1998). Bacteriophage genomic termini were determined via analysis of read start pileups and coverage as previously described (Russell, 2018). Sequencing and assembly results are presented in Table 1.

Annotations were completed using the following programs: DNA Master v2.53.6 (http://cobamide2.bio.pitt.edu/computer.htm), Starterator v1.2 (https://github.com/SEA-PHAGES/starterator), Phamerator v2.5 (Actino_Draft database, https://phamerator.org/) (Cresawn et al., 2011), PhagesDB BLAST (database: actinobacteriophage database; https://phagesdb.org) (Russell and Hatfull, 2017), NCBI BLAST (Databases: blastn standard core nucleotide and blastp standard non-redundant) (Altschul et al., 1990), PECAAN (Rinehart et al., 2016), GeneMark v2.5p (Besemer and Borodovsky, 2005), Glimmer v3.02 (Delcher et al., 2007), Aragorn (v1.1 and v1.2.38) (Laslett and Canback, 2004), HHPRED (Databases: PDB, NCBI-CD, PFam-A, and SCOPe) (Gabler et al., 2020), tRNAscan-SE v2.0 (Lowe and Chan, 2016), DeepTMHMM v1.0.24 (Hallgren et al., 2022), all using the default settings.

MajinBuu has 92 predicted genes that are transcribed unidirectionally, and 26 of these genes were assigned putative functions. Coldpizza has 79 genes, and 31 were assigned putative functions. MajinBuu and Coldpizza were assigned to clusters DJ and DR, respectively, based on gene content similarity of at least 35% to the phages in the Actinobacteriophage database, phagesdb (https://phageDB.org) (Pope et al., 2017; Russell and Hatfull, 2017). As with other cluster DJ phages, genes associated with lysis in MajinBuu, including lysA, lysB, and those encoding holin, are flanked by a conserved set of genes encoding proteins of unknown function that contain transmembrane domains. The proximity and synteny of these transmembrane-containing proteins to lysis genes suggests involvement in lysis and may define a lysis cassette (Bendele et al., 2025). Unlike all cluster DR phages, where both lysA and lysB genes could be identified, no lysB could be identified for Coldpizza. Where Lysin A is typically essential for phage replication, Lysin B is not (Payne et al., 2009). Both genomes do not contain any predicted tRNA or tmRNA genes, which is typical for both clusters. Similarly, neither phage is predicted to be lysogenic due to the absence of identifiable immunity repressor or integrase genes.

Data Availability. MajinBuu GenBank and SRA accession numbers are PV915850 and SRR34858428, respectively. Coldpizza GenBank and SRA accession numbers are PV915803 and SRR34858419, respectively.

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Genome Sequences of Gordonia rubripertincta Bacteriophages MajinBuu and Coldpizza

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