microPublication Biology 2578-9430 Caltech Library 10.17912/micropub.biology.001483 new finding genome announcements bacteriophage Genome Sequence of Microbacterium foliorum Phage KingKamren Gleichsner Alyssa Conceptualization Data curation Formal analysis Methodology Project administration Supervision Validation Writing - original draft Writing - review & editing 1 § Harden Kamren Data curation Investigation Methodology Conceptualization Visualization 1 Salphine Kathryn Data curation Investigation Methodology Validation 1 Sandel Jill Data curation Investigation Methodology Validation 1 Bova Mikaela Data curation Investigation Methodology Validation 1 Denapole Bella Data curation Investigation Methodology Validation 1 Godlewski Anastasia Data curation Investigation Methodology Validation 1 Acevedo Samara Data curation Investigation Methodology Validation 1 Galarneau Alexander Data curation Investigation Methodology Validation 1 Zales Mazon Data curation Investigation Methodology Validation 1 Twumasi Gustavia Data curation Investigation Methodology Validation 1 Voss Sophia Data curation Investigation Methodology Validation 1 Haynes Faith Data curation Investigation Methodology Validation 1 Abdul-Wahhab Rebekah Data curation Investigation Methodology Validation 1 Bu Banfy Data curation Investigation Methodology Validation 1 Favro Abigail Data curation Investigation Methodology Validation 1 Zergaw Amen Data curation Investigation Methodology Validation 1 Maisha Meherun Data curation Investigation Methodology Validation 1 Oliva Bian Investigation Methodology Validation 1 Kaur Sukhpreet Data curation Investigation Validation 1 Kwatia Amma Validation Investigation 1 Rufino Ashley Investigation Validation 1 Arzu Cesia Validation 1 Tyrrell Luke Visualization Writing - original draft 1 Pena Pamela Validation 1 Valentine Megan Data curation Project administration Writing - original draft Conceptualization 1 SUNY Plattsburgh, Plattsburgh, New York, United States Correspondence to: Alyssa Gleichsner ( aglei002@plattsburgh.edu )

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

 10 4 2025 2025 2025 10.17912/micropub.biology.001483 22 12 2024 17 3 2025 28 3 2025 Copyright: © 2025 by the authors 2025 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We report the discovery and genome sequence of a cluster EK bacteriophage, KingKamren, isolated from a soil sample collected in Plattsburgh, New York using the bacteria Microbacterium foliorum , B-24224. Its 54,721 bp genome contains 51 putative genes, 17 of which have predicted functions.

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(A) Plaques of phage KingKamren in top agar with M. foliorum . (B) Gene Content Similarity map, containing GSC values from the GCS tool (Hirokawa et al., 1998) using random selection of 10 phages from the EK1 and EK2 subclusters and all current EK cluster phages. (C) Genome map for KingKamren, with putative genes presented as colored boxes along a genome ruler, in kilobases. Boxes above and below the ruler represent genes that are transcribed rightwards and leftwards, respectively.

Description

Bacteriophages are some of the most genetically diverse entities known (Pope et al., 2015). Understanding this diversity has implications for both ecological and health applications (Milhaven et al., 2023; Strathdee et al., 2023). Here we present the genome of a new bacteriophage, KingKamren, which was collected in 2023 from Plattsburgh NY (44.69179 N, 73.46351 W) using the bacterial host Microbacterium foliorum , B-24224.

Following standard procedures (Zorawik et al., 2024), approximately 15 cm 3 of soil was suspended in 35 mL PYCa medium and shaken at 37°C at 250 rpm for 2 hours followed by centrifugation at 2,000 g and vacuum filtration (0.22 μm filter) of the supernatant. This filtrate was inoculated with M. foliorum and incubated at 30°C for 5 days at 250 rpm. An aliquot was spun at 14,000 g, filtered, and plated in PYCa top agar containing M. foliorum . After 48 hours, KingKamren formed small, turbid plaques with an average size of 1.17 mm (∓ 0.10 SE) in diameter, as determined through measurements using ImageJ (Schnieder et al., 2012) ( Fig. 1A ) and was purified through two rounds of plating.

DNA was isolated from a lysate (Wizard DNA Clean-up Kit, Promega), prepped for sequencing (NEBNext Ultrall FS Kit), sequenced (Illumina sequencing, v3 reagents) and assembled as described by Russell (Russell, 2018). Sequencing resulted in 2,929,352 single-end 150-bp reads with 4,991-fold coverage. The genome was assembled using Newbler v2.9 (Margulies et al,. 2005) and checked for completeness and genome termini using Consed v29.0 (Gordon et al., 1998). Default settings were used unless otherwise noted. This resulted in a genome 54,721 bp in length with 203-bp direct terminal repeat ends and a GC content of 57.5%.

Using standard procedures (Pope et al., 2017), the software DNA Master v5.23.6 (http://cobamide2.bio.pitt.edu), PECAAN (https://discover.kbrinsgd.org), Genemark v2.5p (Lukashin and Borodovsky, 1998), and Glimmer v3.02 (Delcher et al., 1999) were used to predict 53 protein-encoding genes. Start sites were determined using Starterator v485 (https://seaphages.org/software/#Starterator) and Blastp v2.13.0 (Altschul et al., 1990) alignments against the Actinobacteriophage protein (Russell and Hatful, 2017) and NCBI non-redundant protein sequences databases (https://blast.ncbi.nlm.nih.gov). No strong evidence for tRNAs was found using Aragorn v1.2.41 (Laslett and Canback, 2004) and tRNAscan-SE v2.0 (Lowe and Eddy, 1997). A total of 14 genes were assigned putative functions using BLASTp v2.13.0 (Altschul et al., 1990), Phamerator (Cresawn et al., 2011), and HHpred (searching against PDB_mmCIF70, SCOPe70, Pfam-A, and NCBI_Conserved_Domains databases) (Söding et al., 2005). deepTMHMM v1.0.24 (Krogh et al., 2001) and SOSUI (Hirokawa et al. 1998) detected an additional 3 genes as membrane proteins. All software used default settings. The annotation is presented in Fig. 1B

KingKamren was assigned to the EK cluster using the GCS tool (Hirokawa et al., 1998), based on having a gene content similarity (GCS) of at least 35% to other EK bacteriophages in the Actinobacteriophage database. The EK cluster currently contains 56 members. The majority of EK phages are placed into one of two subclusters, EK1 or EK2, but KingKamren is one of three phages (to date) that are not sub-classified, as its GCS is similar to both EK1 and EK2 phages (Figure 1b). This small subset of EK phages share 4 genes (KingKamren’s genes 12, 26, 35, and 47 – Fig. 1C ) of unknown function that are unique to this group and do not share significant sequence similarity to any other actinobacteriophage in the database (phagesdb.org). KingKamren also has a 13,452 bp gene (gene 28, Fig. 1B ) which constitutes 24.6% of its entire genome and encodes a 4,483 amino acid protein of unknown function. This feature is found across all members in the EK phage cluster and represents one of the largest genes in actinobacteriophages (Jacobs-Sera et al., 2020).

Nucleotide sequence accession numbers

KingKamren is available at GenBank with Accession No. XPP978791 and Sequence Read Archive (SRA) No. SRX25029057.

This work is made possible through the generous support of the Howard Hughes Medical Institute Science Education Alliance (SEA) program. We thank the SEA program and community for their training and continuous support, including careful review of the genome annotation and manuscript. The Gene Content Similarity map in Figure 1B is made possible by the discovery and annotation of phages from many institutions and we are thankful for their work. We also thank our two reviewers who gave us valuable feedback on the manuscript.

Altschul Stephen F. Gish Warren Miller Webb Myers Eugene W. Lipman David J. 1990 10 1 Basic local alignment search tool Journal of Molecular Biology 215 3 0022-2836 403 410 10.1016/s0022-2836(05)80360-2 Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12:395–395. https://doi.org/10.1186/1471-2105-12-395 Delcher A. 1999 12 1 Improved microbial gene identification with GLIMMER Nucleic Acids Research 27 23 1362-4962 4636 4641 10.1093/nar/27.23.4636 Hirokawa T, Boon-Chieng S, Mitaku S. 1998. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14:378–379. https://doi.org/10.1093/bioinformatics/14.4.378 Gordon David Abajian Chris Green Phil 1998 3 1 Consed: A Graphical Tool for Sequence Finishing Genome Research 8 3 1088-9051 195 202 10.1101/gr.8.3.195 Jacobs-Sera Deborah Abad Lawrence A. Alvey Richard M. Anders Kirk R. Aull Haley G. Bhalla Suparna S. Blumer Lawrence S. Bollivar David W. Bonilla J. Alfred Butela Kristen A. Coomans Roy J. Cresawn Steven G. D'Elia Tom Diaz Arturo Divens Ashley M. Edgington Nicholas P. Frederick Gregory D. Gainey Maria D. Garlena Rebecca A. Grant Kenneth W. Gurney Susan M. R. Hendrickson Heather L. Hughes Lee E. Kenna Margaret A. Klyczek Karen K. Kotturi Hari Mavrich Travis N. McKinney Angela L. Merkhofer Evan C. Moberg Parker Jordan Molloy Sally D. Monti Denise L. Pape-Zambito Dana A. Pollenz Richard S. Pope Welkin H. Reyna Nathan S. Rinehart Claire A. Russell Daniel A. Shaffer Christopher D. Sivanathan Viknesh Stoner Ty H. Stukey Joseph Sunnen C. Nicole Tolsma Sara S. Tsourkas Philippos K. Wallen Jamie R. Ware Vassie C. Warner Marcie H. Washington Jacqueline M. Westover Kristi M. Whitefleet-Smith JoAnn L. Wiersma-Koch Helen I. Williams Daniel C. Zack Kira M. Hatfull Graham F. 2020 6 18 Genomic diversity of bacteriophages infecting Microbacterium spp PLOS ONE 15 6 1932-6203 e0234636 e0234636 10.1371/journal.pone.0234636 Krogh A, Larsson B, von Heijne G, Sonnhammer EL. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. https://doi.org/10.1006/jmbi.2000.4315 Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16. https://doi.org/10.1093/nar/gkh152 Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964. https://doi.org/10.1093/nar/25.5.955 Lukashin A. 1998 2 15 GeneMark.hmm: new solutions for gene finding Nucleic Acids Research 26 4 1362-4962 1107 1115 10.1093/nar/26.4.1107 Margulies Marcel Egholm Michael Altman William E. Attiya Said Bader Joel S. Bemben Lisa A. Berka Jan Braverman Michael S. Chen Yi-Ju Chen Zhoutao Dewell Scott B. Du Lei Fierro Joseph M. Gomes Xavier V. Godwin Brian C. He Wen Helgesen Scott Ho Chun He Irzyk Gerard P. Jando Szilveszter C. Alenquer Maria L. I. Jarvie Thomas P. Jirage Kshama B. Kim Jong-Bum Knight James R. Lanza Janna R. Leamon John H. Lefkowitz Steven M. Lei Ming Li Jing Lohman Kenton L. Lu Hong Makhijani Vinod B. McDade Keith E. McKenna Michael P. Myers Eugene W. Nickerson Elizabeth Nobile John R. Plant Ramona Puc Bernard P. Ronan Michael T. Roth George T. Sarkis Gary J. Simons Jan Fredrik Simpson John W. Srinivasan Maithreyan Tartaro Karrie R. Tomasz Alexander Vogt Kari A. Volkmer Greg A. Wang Shally H. Wang Yong Weiner Michael P. Yu Pengguang Begley Richard F. Rothberg Jonathan M. 2005 7 31 Genome sequencing in microfabricated high-density picolitre reactors Nature 437 7057 0028-0836 376 380 10.1038/nature03959 Milhaven Mark Versoza Cyril J. Garg Aman Cai Lindsey Cherian Sanjana Johnson Kamalei Salas Perez Kevin Blanco Madison Lobatos Jackelyn Mitra Corinne Strasser Maria Pfeifer Susanne P. 2023 1 10 Microbacterium Cluster EA Bacteriophages: Phylogenomic Relationships and Host Range Predictions Microorganisms 11 1 2076-2607 170 170 10.3390/microorganisms11010170 Pope Welkin H Bowman Charles A Russell Daniel A Jacobs-Sera Deborah Asai David J Cresawn Steven G Jacobs William R Hendrix Roger W Lawrence Jeffrey G Hatfull Graham F Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Phage Hunters Integrating Research and Education Mycobacterial Genetics Course 2015 4 28 Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity eLife 4 2050-084X 10.7554/elife.06416 Pope WH, Jacobs-Sera D, Russell DA, Cresawn SG, Hatfull GF. 2017. SEA-PHAGES Bioinformatics Guide. Howard Hughes Medical Institute, Chevy Chase, MD. Russell DA 2018 Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods Mol Biol 1681 1064-3745 109 125 10.1007/978-1-4939-7343-9_9 29134591 Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database. Bioinformatics 33:784–786. https://doi.org/10.1093/bioinformatics/btw711 Schneider Caroline A Rasband Wayne S Eliceiri Kevin W 2012 6 28 NIH Image to ImageJ: 25 years of image analysis Nature Methods 9 7 1548-7091 671 675 10.1038/nmeth.2089 Söding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244–W248. https://doi.org/10.1093/nar/gki408 Strathdee SA Hatfull GF Mutalik VK Schooley RT 2023 1 5 Phage therapy: From biological mechanisms to future directions. Cell 186 1 0092-8674 17 31 10.1016/j.cell.2022.11.017 36608652 Zorawik M Jacobs-Sera D Freise AC SEA-PHAGES Reddi K 2024 Isolation of Bacteriophages on Actinobacteria Hosts. Methods Mol Biol 2793 1064-3745 273 298 10.1007/978-1-0716-3798-2_17 38526736