{ "componentChunkName": "component---src-templates-article-page-js", "path": "/journals/biology/micropub-biology-002002", "result": {"data":{"article":{"manuscript":{"id":"4e039532-c8ea-4721-b3a3-5c3310abfd21","submissionTypes":["new finding"],"citations":[],"doi":"10.17912/micropub.biology.002002","dbReferenceId":"","pmcId":"","pmId":"","proteopedia":"","reviewPanel":"","species":["bacteriophage"],"integrations":[],"corrections":null,"history":{"received":"2025-12-23T14:40:03.137Z","revisionReceived":"2026-07-09T18:17:09.630Z","accepted":"2026-08-19T21:50:35.831Z","published":"2026-09-17T15:30:07.776Z","indexed":"2026-10-01T15:30:07.776Z"},"versions":[{"id":"4877c03d-18e5-442c-96dc-dd4569ea025b","decision":"revise","abstract":"

Wrackline is a novel bacteriophage isolated from sand collected along the high tide line and beneath organic material on Willard Beach, ME. It was isolated using Microbacterium sp. strain Casco Bay, a marine bacterium, and is also capable of infecting Microbacterium foliorum, a bacterium originally isolated from grass. Wrackline's genome is 42,148 bp and encodes 80 putative protein coding genes, 26 of which have no known homologs. Based on gene content similarity, Wrackline is assigned to actinobacteriophage cluster GF.

","acknowledgements":"

We would like to thank Sam R. Cousins for the discovery, naming, and isolation of Wrackline. Thanks as well to Kai Watkins for their isolation of Microbacterium sp. Casco Bay and for their assistance. Thank you to the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for the electron microscopy image. A special thanks to Sally Molloy for the annotation review and guidance. We are grateful for sequencing done by Dan Russell, Becky Garlena, and Deborah Jacobs-Sera and for their guidance through the annotation process. Thanks as well to Vic Sivanathan and the Howard Hughes Medical Institute (HHMI) along with Dr. Graham Hatfull and the SEA-PHAGES program for their support in our research.  

","authors":[{"affiliations":["Southern Maine Community College"],"departments":["Biology"],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"kmwprints@gmail.com","firstName":"Kit","lastName":"Williams","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0000-3864-6059"},{"affiliations":["Southern Maine Community College"],"departments":["Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"esavage@mainecc.edu","firstName":"Emily","lastName":"Savage","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0009-0006-0316-2277"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.

","image":{"url":"https://portal.micropublication.org/uploads/62d36d5ed3e73902b28c2d636bfccede.png"},"imageCaption":"

Left:Transmission electron micrograph of bacteriophage Wrackline displaying a siphovirus morphology (Hitachi HT7800, 120kV, accelerating voltage 100kV) using negative stain (1% uranyl acetate). Wrackline has an icosahedral capsid 60nm in diameter and a tail of 150nm in length (n=1). Scale bar = 200 nm. Right: Spot dilution assay of phage Wrackline (bottom of plates) and Cluster EE phage Gardevoir (top of plates) plated with Microbacterium sp. Casco Bay on Marine agar (left plate) or  with M. foliorum plated on PYCa (right plate).  Wrackline shows similar plating efficiency on both bacterial hosts.

","imageTitle":"

TEM imaging and plaques of bacteriophage Wrackline

","methods":"

","reagents":"

","patternDescription":"

Gram-positive marine bacteria are understudied and typically challenging to culture (Gontang et al., 2007; Watkins 2022). Microbacterium sp. Casco Bay is halotolerant and simple to culture, making it an ideal host for the isolation and characterization of marine bacteriophage (Girard et al., 2025). While the whole genome of M. sp. Casco Bay has not yet been sequenced, 16s rRNA gene sequencing categorized it as a microbacterial strain.

Bacteriophage Wrackline was isolated from a sand sample taken just above the high tide line on Willard Beach, South Portland, Maine (Global Positioning System [GPS] 43.645167 N, -70.227596 W). The phage was isolated using standard procedures (Zorawik et al., 2024).  Marine broth (75% filtered sea water, yeast extract, peptone, casamino acids, and glycerin) inoculated with M. sp. Casco Bay and five grams of sand were combined and incubated for 4 days at 30°C. The culture was filtered (0.22 μm pore size) and the filtrate spotted on to solidified marine top agar (yeast extract, peptone, casamino acids, and 5g/L of agar) supplemented with M. sp. Casco Bay on a plate of marine agar (MA) (75% filtered sea water, yeast extract, peptone, and 15g/L of agar). After 48 hours at 30°C, Wrackline formed plaques with a clear center approximately 0.4 – 0.6mm wide surrounded by a 0.2 – 0.4mm turbid halo (n=5) . Negative stain transmission electron microscopy showed siphovirus morphology with capsid size of 60nm and a 150nm long tail (n=1)(Fig 1).

DNA was extracted from a Wrackline lysate using the Promega Wizard DNA clean-up kit. Sequencing library preparation was done using the NEB Ultra II Library Kit, and the library sequenced on the Illumina NextSeq 1000 sequencer with the XLEAP-PI kit. This yielded 2.6 million 100 base single-end reads, providing 5,885-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 argument (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014) and genomic termini and completeness was checked using Consed V29 (Gordon and Green, 2013) default parameters. Wrackline's genome is 42,148 bp with a 69.2% GC content and is circularly permuted.

Wrackline was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017) using Glimmer (v3.02b) (Delcher et al., 2007) and GeneMark (v2.5p) (Besemer and Borodovsky, 2005) to assess coding potential and identify putative genes. Start sites were then refined and gene functions predicted using Phamerator (Actino_Draft v597) (Cresawn et al., 2011), Starterator (v3.02) (Pacey, 2016), HHPred (v2.08 (PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD]) (Söding et al., 2005), and Blastp (Actinobacteriophage proteins, non-redundant protein sequences [nr]) (Altschul et al., 1990). DeepTMHMM (v1.2.33.c.) (Hallgren et al., 2022) and SOSUI (v1.11) (Hirokawa et al., 1998) were used to assess the presence of transmembrane proteins. Default parameters were used for all software programs.

This analysis identified 80 predicted protein-coding genes, 26 of which are orphams with no known homologs (Cresawn et al., 2011).

Cluster assignment based on gene content similarity (GCS) of at least 35% to other phages in the Actinobacteria database (PhagesDB) placed Wrackline in cluster GF which only contains 3 other phages to date (Pope et al., 2017; Russell and Hatfull, 2017). The other cluster GF phages share more than 76.3% GCS, while Wrackline stands out sharing only 48.6% GCS with the group. Additionally, the other phages in the cluster were isolated using Microbacterium foliorum. Wrackline is also able to infect M. foliorum, with similar plaquing efficiency on both strains (Fig 1).

Nucleotide sequence accession numbers

Wrackline is available at GenBank with Accession No. PV876983 and Sequence Read Archive (SRA) No. SRX29714291. The host strain Microbacterium sp. Casco Bay is archived in the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Science (B81).

","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":"

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":"

Girard LR, Cousins SR, Heald AC, Tanzey DA, Omo EM, Flannigan CR, et al., Savage. 2025. Genome sequence of bacteriophage PensacolaC28 isolated using\n Microbacterium sp\n . Casco Bay. Microbiology Resource Announcements 14: 10.1128/mra.01146-24.

","pubmedId":"","doi":"10.1128/mra.01146-24"},{"reference":"

Gontang EA, Fenical W, Jensen PR. 2007. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments. Applied and Environmental Microbiology 73: 3272-3282.

","pubmedId":"","doi":"10.1128/AEM.02811-06"},{"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":"

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":"

Lleò MdM, Signoretto C, Canepari P. Undefined. Gram-Positive Bacteria in the Marine Environment. Oceans and Health: Pathogens in the Marine Environment : 307-330.

","pubmedId":"","doi":"10.1007/0-387-23709-7_13"},{"reference":"

Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.

","pubmedId":"","doi":"doi.org/10.14806/ej.17.1.200"},{"reference":"

Pacey M. 2016. Edited by Pope W. Starterator Guide. University of Pittsburgh, Pittsburgh, PA. Available from: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf

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

Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"

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":"

Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.

","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"

Watkins K. 2022. The Effects of Prophage Integration in a New Bacterial Host. https://doi.org/10.13140/RG.2.2.28243.39203

","pubmedId":"","doi":"10.13140/RG.2.2.28243.39203"},{"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 Sequence of Novel Cluster GF Bacteriophage Wrackline, Isolated on Microbacterium sp. Casco Bay

","reviews":[{"reviewer":{"displayName":"Sara Tolsma"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Nic Vega"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"53fe2350-f8d5-417a-a464-d304851ab705","decision":"revise","abstract":"

Wrackline is a novel bacteriophage isolated from sand collected along the high tide line and beneath organic material on Willard Beach, ME. It was isolated using Microbacterium sp. strain Casco Bay, a marine bacterium, and is also capable of infecting Microbacterium foliorum, a bacterium originally isolated from grass. Wrackline's genome is 42,148 bp and encodes 80 putative protein coding genes, 26 of which have no known homologs. Based on gene content similarity, Wrackline is assigned to actinobacteriophage cluster GF.

","acknowledgements":"

We would like to thank Sam R. Cousins for the discovery, naming, and isolation of Wrackline. Thanks as well to Kai Watkins for their isolation of Microbacterium sp. Casco Bay and for their assistance. Thank you to the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for the electron microscopy image. A special thanks to Sally Molloy for the annotation review and guidance. We are grateful for sequencing done by Dan Russell, Becky Garlena, and Deborah Jacobs-Sera and for their guidance through the annotation process. Thanks as well to Vic Sivanathan and the Howard Hughes Medical Institute (HHMI) along with Dr. Graham Hatfull and the SEA-PHAGES program for their support in our research.  

","authors":[{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology",""],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"kmwprints@gmail.com","firstName":"Kit","lastName":"Williams","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0000-3864-6059"},{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"esavage@mainecc.edu","firstName":"Emily","lastName":"Savage","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0009-0006-0316-2277"}],"awards":[],"conflictsOfInterest":"

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

","dataTable":{"url":"https://portal.micropublication.org/uploads/6d6c75a233567797669178010fea5c4d.csv"},"extendedData":[],"funding":"

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.

","image":{"url":"https://portal.micropublication.org/uploads/9b29c04ed3dccc206c62e0c4ead66bae.jpeg"},"imageCaption":"

Figure 1: A: Bacteriophage Wrackline forms clear plaques approximately 0.4 – 0.6mm in diameter (n=5). B: Transmission electron micrograph of bacteriophage Wrackline displaying a siphovirus morphology (Hitachi HT7800, 120kV, accelerating voltage 100kV) using negative stain (1% uranyl acetate). Wrackline has an icosahedral capsid 60nm in diameter and a tail of 150nm in length (n=1). Scale bar = 200 nm. C: Spot dilution assay of phage Wrackline and Cluster EE phage Gardevoir ( + control) plated with M. foliorum plated on PYCa, and Microbacterium sp. Casco Bay on Marine agar. Dilutions 10-5 through 10-7 show Wrackline shows similar plating efficiency on both bacterial hosts. 

","imageTitle":"

Plaques, TEM image, and host range assessment of Bacteriophage Wrackline

","methods":"

","reagents":"

","patternDescription":"

Gram-positive marine bacteria are understudied and typically challenging to culture (Gontang et al., 2007; Watkins 2022). Microbacterium sp. Casco Bay is halotolerant and simple to culture, making it an ideal host for the isolation and characterization of marine bacteriophage (Girard et al., 2025). While the whole genome of M. sp. Casco Bay has not yet been sequenced, 16S rRNA gene sequencing categorized it as a microbacterial strain. 

Bacteriophage Wrackline was isolated from a sand sample taken just above the high tide line on Willard Beach, South Portland, Maine (Global Positioning System [GPS] 43.645167 N, -70.227596 W). The phage was isolated using standard procedures (Zorawik et al., 2024).  Marine broth (Table 1) inoculated with M. sp. Casco Bay and five grams of sand were combined and incubated for 4 days at 30°C. The culture was filtered (0.22 μm pore size) and the filtrate spotted on to solidified marine top agar (Table 1) supplemented with M. sp. Casco Bay on a plate of marine agar (MA) (Table 1). After 48 hours at 30°C, Wrackline formed plaques with a clear center approximately 0.4–0.6mm wide (n=5) (Fig 1. A). Negative stain transmission electron microscopy showed siphovirus morphology with capsid size of 60nm and a 150nm long tail (n=1) (Fig 1. B). 

DNA was extracted from a Wrackline lysate using the Promega Wizard DNA clean-up kit. Sequencing library preparation was done using the NEB Ultra II Library Kit, and the library sequenced on the Illumina NextSeq 1000 sequencer with the XLEAP-PI kit. This yielded 2.6 million 100 base single-end reads, providing 5,885-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 argument (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014) and genomic termini and completeness was checked using Consed V29 (Gordon and Green, 2013) default parameters. Wrackline's genome is 42,148 bp with a 69.2% GC content and is circularly permuted. 

Wrackline was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017) using Glimmer (v3.02b) (Delcher et al., 2007) and GeneMark (v2.5p) (Besemer and Borodovsky, 2005) to assess coding potential and identify putative genes. Start sites were then refined and gene functions predicted using Phamerator (Actino_Draft v597) (Cresawn et al., 2011), Starterator (v3.02) (Pacey, 2016), HHPred (v2.08 (PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD]) (Söding et al., 2005), and Blastp (Actinobacteriophage database, NCBI non-redundant database) (Altschul et al., 1990). DeepTMHMM (v1.2.33.c.) (Hallgren et al., 2022) and SOSUI (v1.11) (Hirokawa et al., 1998) were used to assess the presence of transmembrane proteins. No tRNA genes were detected using Aragorn (tRNA-scan SE v1.2.41 v). Default parameters were used for all software programs. 

This analysis identified 80 predicted protein-coding genes, 26 of which are orphams with no known homologs (Cresawn et al., 2011). The left arm of the genome is transcribed in the forward orientation and comprised of structural genes and the endolysin, followed by a reverse transcribed region containing genes of unknown functions and DNA binding domains, then returning to the forward orientation. The right arm of the genome contains a RecE-like exonuclease and RecT-like DNA pairing protein (GP 64 and 65) which may work together as a recombinase.  

Cluster assignment based on gene content similarity (GCS) of at least 35% to other phages in the Actinobacteria database (Actino_Draft database) placed Wrackline in cluster GF which only contains 3 other phages to date (Pope et al., 2017; Russell and Hatfull, 2017). The other cluster GF phages share more than 76.3% GCS, while Wrackline stands out sharing only 48.6% GCS with the group. While the structural genes of Wrackline are similar to other GF phages, the first 12 genes vary from others in the cluster and among those are 8 orphams for which no function was assigned. The reverse section of the genome from GP 38 – 60 shares some protein phams with other GFs, but is an orpham rich region, with 7 orphams with no functional assignment.  Additionally, other phages in the cluster were isolated using Microbacterium foliorum. Host range for phage Wrackline was evaluated by standard procedures (Zorawik et al., 2024). Spot plates were prepared using Wrackline and a control phage (Gardevoir (EE)) known to infect Microbacterium foliorum and Microbacterium sp. Casco Bay. Phage lysate for Wrackline and Gardevoir were diluted to 10-7 and 2uL were spotted onto each host. Wrackline is able to infect M. foliorum with a similar plating efficiency (Fig 1. C).  

Nucleotide sequence accession numbers

Wrackline is available at GenBank with Accession No. PV876983 and Sequence Read Archive (SRA) No. SRX29714291. The host strain Microbacterium sp. Casco Bay is archived in the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Science (B81).

","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":"

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":"

Girard LR, Cousins SR, Heald AC, Tanzey DA, Omo EM, Flannigan CR, et al., Savage. 2025. Genome sequence of bacteriophage PensacolaC28 isolated using\n Microbacterium sp\n . Casco Bay. Microbiology Resource Announcements 14: 10.1128/mra.01146-24.

","pubmedId":"","doi":"10.1128/mra.01146-24"},{"reference":"

Gontang EA, Fenical W, Jensen PR. 2007. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments. Applied and Environmental Microbiology 73: 3272-3282.

","pubmedId":"","doi":"10.1128/AEM.02811-06"},{"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":"

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":"

Lleò MdM, Signoretto C, Canepari P. Undefined. Gram-Positive Bacteria in the Marine Environment. Oceans and Health: Pathogens in the Marine Environment : 307-330.

","pubmedId":"","doi":"10.1007/0-387-23709-7_13"},{"reference":"

Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.

","pubmedId":"","doi":"doi.org/10.14806/ej.17.1.200"},{"reference":"

Pacey M. 2016. Edited by Pope W. Starterator Guide. University of Pittsburgh, Pittsburgh, PA. Available from: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf

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

Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"

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":"

Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.

","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"

Watkins K. 2022. The Effects of Prophage Integration in a New Bacterial Host. https://doi.org/10.13140/RG.2.2.28243.39203

","pubmedId":"","doi":"10.13140/RG.2.2.28243.39203"},{"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 Sequence of Novel Cluster GF Bacteriophage Wrackline, Isolated on Microbacterium sp. Casco Bay

","reviews":[],"curatorReviews":[]},{"id":"c923a147-021f-42ea-b5d9-d19134dae510","decision":"revise","abstract":"

Wrackline is a novel bacteriophage isolated from sand collected along the high tide line and beneath organic material on Willard Beach, ME. It was isolated using Microbacterium sp. strain Casco Bay, a marine bacterium, and is also capable of infecting Microbacterium foliorum, a bacterium originally isolated from grass. Wrackline's genome is 42,148 bp and encodes 80 putative protein coding genes, 26 of which have no known homologs. Based on gene content similarity, Wrackline is assigned to actinobacteriophage cluster GF.

","acknowledgements":"

We would like to thank Sam R. Cousins for the discovery, naming, and isolation of Wrackline. Thanks as well to Kai Watkins for their isolation of Microbacterium sp. Casco Bay and for their assistance. Thank you to the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for the electron microscopy image. A special thanks to Sally Molloy for the annotation review and guidance. We are grateful for sequencing done by Dan Russell, Becky Garlena, and Deborah Jacobs-Sera and for their guidance through the annotation process. Thanks as well to Vic Sivanathan and the Howard Hughes Medical Institute (HHMI) along with Dr. Graham Hatfull and the SEA-PHAGES program for their support in our research.  

","authors":[{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology",""],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"kmwprints@gmail.com","firstName":"Kit","lastName":"Williams","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0000-3864-6059"},{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"esavage@mainecc.edu","firstName":"Emily","lastName":"Savage","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0009-0006-0316-2277"}],"awards":[],"conflictsOfInterest":"

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

","dataTable":{"url":null},"extendedData":[],"funding":"

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.

","image":{"url":"https://portal.micropublication.org/uploads/9b29c04ed3dccc206c62e0c4ead66bae.jpeg"},"imageCaption":"

Figure 1: A: Bacteriophage Wrackline forms clear plaques approximately 0.4 – 0.6mm in diameter (n=5). B: Transmission electron micrograph of bacteriophage Wrackline displaying a siphovirus morphology (Hitachi HT7800, 120kV, accelerating voltage 100kV) using negative stain (1% uranyl acetate). Wrackline has an icosahedral capsid 60nm in diameter and a tail of 150nm in length (n=1). Scale bar = 200 nm. C: Spot dilution assay of phage Wrackline and Cluster EE phage Gardevoir ( + control) plated with M. foliorum plated on PYCa, and Microbacterium sp. Casco Bay on Marine agar. Dilutions 10-5 through 10-7 show Wrackline shows similar plating efficiency on both bacterial hosts. 

","imageTitle":"

Plaques, TEM image, and host range assessment of Bacteriophage Wrackline

","methods":"

","reagents":"

Agar

Ingredients

Marine Agar

1g yeast extract

5g bacto peptone

15g agar

Marine Top Agar

0.5g yeast extract

0.5g bacto peptone

0.5g cas amino acids

5g agar

1mL of 1M CaCL2 per 100mL is added post-autoclave

PYCa Recipies for M. foliorum

PYCa

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

15g agar

1mL cyclohexamide added post-autoclave

PYCa Top Agar

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

7g agar

4.5mL 1M CaCl added post-autoclave

","patternDescription":"

Gram-positive marine bacteria are understudied and typically challenging to culture (Gontang et al., 2007; Watkins 2022). Microbacterium sp. Casco Bay is halotolerant and simple to culture, making it an ideal host for the isolation and characterization of marine bacteriophage (Girard et al., 2025). While the whole genome of M. sp. Casco Bay has not yet been sequenced, 16S rRNA gene sequencing categorized it as a microbacterial strain. 

Bacteriophage Wrackline was isolated from a sand sample taken just above the high tide line on Willard Beach, South Portland, Maine (Global Positioning System [GPS] 43.645167 N, -70.227596 W). The phage was isolated using standard procedures (Zorawik et al., 2024).  Marine broth (Table 1) inoculated with M. sp. Casco Bay and five grams of sand were combined and incubated for 4 days at 30°C. The culture was filtered (0.22 μm pore size) and the filtrate spotted on to solidified marine top agar (Table 1) supplemented with M. sp. Casco Bay on a plate of marine agar (MA) (Table 1). After 48 hours at 30°C, Wrackline formed plaques with a clear center approximately 0.4–0.6mm wide (n=5) (Fig 1. A). Negative stain transmission electron microscopy showed siphovirus morphology with capsid size of 60nm and a 150nm long tail (n=1) (Fig 1. B). 

DNA was extracted from a Wrackline lysate using the Promega Wizard DNA clean-up kit. Sequencing library preparation was done using the NEB Ultra II Library Kit, and the library sequenced on the Illumina NextSeq 1000 sequencer with the XLEAP-PI kit. This yielded 2.6 million 100 base single-end reads, providing 5,885-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 argument (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014) and genomic termini and completeness was checked using Consed V29 (Gordon and Green, 2013) default parameters. Wrackline's genome is 42,148 bp with a 69.2% GC content and is circularly permuted. 

Wrackline was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017) using Glimmer (v3.02b) (Delcher et al., 2007) and GeneMark (v2.5p) (Besemer and Borodovsky, 2005) to assess coding potential and identify putative genes. Start sites were then refined and gene functions predicted using Phamerator (Actino_Draft v597) (Cresawn et al., 2011), Starterator (v3.02) (Pacey, 2016), HHPred (v2.08 (PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD]) (Söding et al., 2005), and Blastp (Actinobacteriophage database, NCBI non-redundant database) (Altschul et al., 1990). DeepTMHMM (v1.2.33.c.) (Hallgren et al., 2022) and SOSUI (v1.11) (Hirokawa et al., 1998) were used to assess the presence of transmembrane proteins. No tRNA genes were detected using Aragorn (tRNA-scan SE v1.2.41 v). Default parameters were used for all software programs. 

This analysis identified 80 predicted protein-coding genes, 26 of which are orphams with no known homologs (Cresawn et al., 2011). The left arm of the genome is transcribed in the forward orientation and comprised of structural genes and the endolysin, followed by a reverse transcribed region containing genes of unknown functions and DNA binding domains, then returning to the forward orientation. The right arm of the genome contains a RecE-like exonuclease and RecT-like DNA pairing protein (GP 64 and 65) which may work together as a recombinase.  

Cluster assignment based on gene content similarity (GCS) of at least 35% to other phages in the Actinobacteria database (Actino_Draft database) placed Wrackline in cluster GF which only contains 3 other phages to date (Pope et al., 2017; Russell and Hatfull, 2017). The other cluster GF phages share more than 76.3% GCS, while Wrackline stands out sharing only 48.6% GCS with the group. While the structural genes of Wrackline are similar to other GF phages, the first 12 genes vary from others in the cluster and among those are 8 orphams for which no function was assigned. The reverse section of the genome from GP 38 – 60 shares some protein phams with other GFs, but is an orpham rich region, with 7 orphams with no functional assignment.  Additionally, other phages in the cluster were isolated using Microbacterium foliorum. Host range for phage Wrackline was evaluated by standard procedures (Zorawik et al., 2024). Spot plates were prepared using Wrackline and a control phage (Gardevoir (EE)) known to infect Microbacterium foliorum and Microbacterium sp. Casco Bay. Phage lysate for Wrackline and Gardevoir were diluted to 10-7 and 2uL were spotted onto each host. Wrackline is able to infect M. foliorum with a similar plating efficiency (Fig 1. C).  

Nucleotide sequence accession numbers

Wrackline is available at GenBank with Accession No. PV876983 and Sequence Read Archive (SRA) No. SRX29714291. The host strain Microbacterium sp. Casco Bay is archived in the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Science (B81).

","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":"

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":"

Girard LR, Cousins SR, Heald AC, Tanzey DA, Omo EM, Flannigan CR, et al., Savage. 2025. Genome sequence of bacteriophage PensacolaC28 isolated using\n Microbacterium sp\n . Casco Bay. Microbiology Resource Announcements 14: 10.1128/mra.01146-24.

","pubmedId":"","doi":"10.1128/mra.01146-24"},{"reference":"

Gontang EA, Fenical W, Jensen PR. 2007. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments. Applied and Environmental Microbiology 73: 3272-3282.

","pubmedId":"","doi":"10.1128/AEM.02811-06"},{"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":"

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":"

Lleò MdM, Signoretto C, Canepari P. Undefined. Gram-Positive Bacteria in the Marine Environment. Oceans and Health: Pathogens in the Marine Environment : 307-330.

","pubmedId":"","doi":"10.1007/0-387-23709-7_13"},{"reference":"

Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.

","pubmedId":"","doi":"doi.org/10.14806/ej.17.1.200"},{"reference":"

Pacey M. 2016. Edited by Pope W. Starterator Guide. University of Pittsburgh, Pittsburgh, PA. Available from: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf

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

Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"

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":"

Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.

","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"

Watkins K. 2022. The Effects of Prophage Integration in a New Bacterial Host. https://doi.org/10.13140/RG.2.2.28243.39203

","pubmedId":"","doi":"10.13140/RG.2.2.28243.39203"},{"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 Sequence of Novel Cluster GF Bacteriophage Wrackline, Isolated on Microbacterium sp. Casco Bay

","reviews":[{"reviewer":{"displayName":"Nic Vega"},"openAcknowledgement":true,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"49bfcfa2-7190-4dd4-9a59-54403fa59240","decision":"accept","abstract":"

Wrackline is a novel bacteriophage isolated from sand collected along the high tide line and beneath organic material on Willard Beach, ME. It was isolated using Microbacterium sp. strain Casco Bay, a marine bacterium, and is also capable of infecting Microbacterium foliorum, a bacterium originally isolated from grass. Wrackline's genome is 42,148 bp and encodes 80 putative protein coding genes, 26 of which have no known homologs. Based on gene content similarity, Wrackline is assigned to actinobacteriophage cluster GF.

","acknowledgements":"

We would like to thank Sam R. Cousins for the discovery, naming, and isolation of Wrackline. Thanks as well to Kai Watkins for their isolation of Microbacterium sp. Casco Bay and for their assistance. Thank you to the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for the electron microscopy image. A special thanks to Sally Molloy for the annotation review and guidance. We are grateful for sequencing done by Dan Russell, Becky Garlena, and Deborah Jacobs-Sera and for their guidance through the annotation process. Thanks as well to Vic Sivanathan and the Howard Hughes Medical Institute (HHMI) along with Dr. Graham Hatfull and the SEA-PHAGES program for their support in our research.  

","authors":[{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology",""],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"kmwprints@gmail.com","firstName":"Kit","lastName":"Williams","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0000-3864-6059"},{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"esavage@mainecc.edu","firstName":"Emily","lastName":"Savage","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0009-0006-0316-2277"}],"awards":[],"conflictsOfInterest":"

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

","dataTable":{"url":null},"extendedData":[],"funding":"

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.

","image":{"url":"https://portal.micropublication.org/uploads/9b29c04ed3dccc206c62e0c4ead66bae.jpeg"},"imageCaption":"

Figure 1: A: Bacteriophage Wrackline forms clear plaques approximately 0.4 – 0.6mm in diameter (n=5). B: Transmission electron micrograph of bacteriophage Wrackline displaying a siphovirus morphology (Hitachi HT7800, 120kV, accelerating voltage 100kV) using negative stain (1% uranyl acetate). Wrackline has an icosahedral capsid 60nm in diameter and a tail of 150nm in length (n=1). Scale bar = 200 nm. C: Spot dilution assay of phage Wrackline and Cluster EE phage Gardevoir ( + control) plated with M. foliorum plated on PYCa, and Microbacterium sp. Casco Bay on Marine agar. Dilutions 10-5 through 10-7 show Wrackline shows similar plating efficiency on both bacterial hosts. 

","imageTitle":"

Plaques, TEM image, and host range assessment of Bacteriophage Wrackline

","methods":"

","reagents":"

Marine Agar Recipies for M. sp. Casco Bay

Marine Broth

750mL filtered seawater

250mL distilled water

0.5g yeast extract

0.5g casamino acids

3mL glycerine

Marine Agar

750mL filtered seawater

250mL distilled water

1g yeast extract

5g bacto peptone

15g agar

Marine Top Agar

750mL filtered seawater

250mL distilled water

0.5g yeast extract

0.5g bacto peptone

0.5g cas amino acids

5g agar

1mL of 1M CaCL2 per 100mL is added post-autoclave

PYCa Recipies for M. foliorum

PYCa Broth

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

PYCa Agar

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

15g agar

1mL cycloheximide (10mg/mL) added post-autoclave

PYCa Top Agar

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

7g agar

1mL cycloheximide (10mg/mL) added post-autoclave

1mL of 1M CaCL2 per 100mL is added post-autoclave

","patternDescription":"

Gram-positive marine bacteria are understudied and typically challenging to culture (Gontang et al., 2007; Watkins 2022). Microbacterium sp. Casco Bay is halotolerant and simple to culture, making it an ideal host for the isolation and characterization of marine bacteriophage (Girard et al., 2025). While the whole genome of M. sp. Casco Bay has not yet been sequenced, 16S rRNA gene sequencing categorized it as a microbacterial strain. 

Bacteriophage Wrackline was isolated from a sand sample taken just above the high tide line on Willard Beach, South Portland, Maine (Global Positioning System [GPS] 43.645167 N, -70.227596 W). The phage was isolated using standard procedures (Zorawik et al., 2024).  Marine broth (Table 1) inoculated with M. sp. Casco Bay and five grams of sand were combined and incubated for 4 days at 30°C. The culture was filtered (0.22 μm pore size) and the filtrate spotted on to solidified marine top agar (Table 1) supplemented with M. sp. Casco Bay on a plate of marine agar (MA) (Table 1). After 48 hours at 30°C, Wrackline formed plaques with a clear center approximately 0.4–0.6mm wide (n=5) (Fig 1. A). Negative stain transmission electron microscopy showed siphovirus morphology with capsid size of 60nm and a 150nm long tail (n=1) (Fig 1. B). 

DNA was extracted from a Wrackline lysate using the Promega Wizard DNA clean-up kit. Sequencing library preparation was done using the NEB Ultra II Library Kit, and the library sequenced on the Illumina NextSeq 1000 sequencer with the XLEAP-PI kit. This yielded 2.6 million 100 base single-end reads, providing 5,885-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 argument (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014) and genomic termini and completeness was checked using Consed V29 (Gordon and Green, 2013) default parameters. Wrackline's genome is 42,148 bp with a 69.2% GC content and is circularly permuted. 

Wrackline was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017) using Glimmer (v3.02b) (Delcher et al., 2007) and GeneMark (v2.5p) (Besemer and Borodovsky, 2005) to assess coding potential and identify putative genes. Start sites were then refined and gene functions predicted using Phamerator (Actino_Draft v597) (Cresawn et al., 2011), Starterator (v3.02) (Pacey, 2016), HHPred (v2.08 (PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD]) (Söding et al., 2005), and Blastp (Actinobacteriophage database, NCBI non-redundant database) (Altschul et al., 1990). DeepTMHMM (v1.2.33.c.) (Hallgren et al., 2022) and SOSUI (v1.11) (Hirokawa et al., 1998) were used to assess the presence of transmembrane proteins. No tRNA genes were detected using Aragorn (tRNA-scan SE v1.2.41 v). Default parameters were used for all software programs. 

This analysis identified 80 predicted protein-coding genes, 26 of which are orphams with no known homologs (Cresawn et al., 2011). The left arm of the genome is transcribed in the forward orientation and comprised of structural genes and the endolysin, followed by a reverse transcribed region containing genes of unknown functions and DNA binding domains, then returning to the forward orientation. The right arm of the genome contains a RecE-like exonuclease and RecT-like DNA pairing protein (GP 64 and 65) which may work together as a recombinase.  

Cluster assignment based on gene content similarity (GCS) of at least 35% to other phages in the Actinobacteria database (Actino_Draft database) placed Wrackline in cluster GF which only contains 3 other phages to date (Pope et al., 2017; Russell and Hatfull, 2017). The other cluster GF phages share more than 76.3% GCS, while Wrackline stands out sharing only 48.6% GCS with the group. While the structural genes of Wrackline are similar to other GF phages, the first 12 genes vary from others in the cluster and among those are 8 orphams for which no function was assigned. The reverse section of the genome from GP 38 – 60 shares some protein phams with other GFs, but is an orpham rich region, with 7 orphams with no functional assignment.  Additionally, other phages in the cluster were isolated using Microbacterium foliorum. Host range for phage Wrackline was evaluated by standard procedures (Zorawik et al., 2024). Spot plates were prepared using Wrackline and a control phage (Gardevoir (EE)) known to infect Microbacterium foliorum and Microbacterium sp. Casco Bay. Phage lysate for Wrackline and Gardevoir were diluted to 10-7 and 2uL were spotted onto each host. Wrackline is able to infect M. foliorum with a similar plating efficiency (Fig 1. C).  

Nucleotide sequence accession numbers

Wrackline is available at GenBank with Accession No. PV876983 and Sequence Read Archive (SRA) No. SRX29714291. The host strain Microbacterium sp. Casco Bay is archived in the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Science (B81).

","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":"

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":"

Girard LR, Cousins SR, Heald AC, Tanzey DA, Omo EM, Flannigan CR, et al., Savage. 2025. Genome sequence of bacteriophage PensacolaC28 isolated using\n Microbacterium sp\n . Casco Bay. Microbiology Resource Announcements 14: 10.1128/mra.01146-24.

","pubmedId":"","doi":"10.1128/mra.01146-24"},{"reference":"

Gontang EA, Fenical W, Jensen PR. 2007. Phylogenetic Diversity of Gram-Positive Bacteria Cultured from Marine Sediments. Applied and Environmental Microbiology 73: 3272-3282.

","pubmedId":"","doi":"10.1128/AEM.02811-06"},{"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":"

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":"

Lleò MdM, Signoretto C, Canepari P. Undefined. Gram-Positive Bacteria in the Marine Environment. Oceans and Health: Pathogens in the Marine Environment : 307-330.

","pubmedId":"","doi":"10.1007/0-387-23709-7_13"},{"reference":"

Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.

","pubmedId":"","doi":"doi.org/10.14806/ej.17.1.200"},{"reference":"

Pacey M. 2016. Edited by Pope W. Starterator Guide. University of Pittsburgh, Pittsburgh, PA. Available from: https://seaphages.org/media/docs/Starterator_Guide_2016.pdf

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

Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.

","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"

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":"

Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.

","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"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":"

Watkins K. 2022. The Effects of Prophage Integration in a New Bacterial Host. https://doi.org/10.13140/RG.2.2.28243.39203

","pubmedId":"","doi":"10.13140/RG.2.2.28243.39203"},{"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 Sequence of Novel Cluster GF Bacteriophage Wrackline, Isolated on Microbacterium sp. Casco Bay

","reviews":[],"curatorReviews":[]},{"id":"1fdf5b21-01d6-43e9-9a69-232e386802f6","decision":"publish","abstract":"

Wrackline is a novel bacteriophage isolated from sand collected along the high tide line and beneath organic material on Willard Beach, ME. It was isolated using Microbacterium sp. strain Casco Bay, a marine bacterium, and is also capable of infecting Microbacterium foliorum, a bacterium originally isolated from grass. Wrackline's genome is 42,148 bp and encodes 80 putative protein coding genes, 26 of which have no known homologs. Based on gene content similarity, Wrackline is assigned to actinobacteriophage cluster GF.

","acknowledgements":"

We would like to thank Sam R. Cousins for the discovery, naming, and isolation of Wrackline. Thanks as well to Kai Watkins for their isolation of Microbacterium sp. Casco Bay and for their assistance. Thank you to the University of Maryland Baltimore County (UMBC) and Tagide deCarvalho at the Keith R. Porter Imaging Facility for the electron microscopy image. A special thanks to Sally Molloy for the annotation review and guidance. We are grateful for sequencing done by Dan Russell, Becky Garlena, and Deborah Jacobs-Sera and for their guidance through the annotation process. Thanks as well to Vic Sivanathan and the Howard Hughes Medical Institute (HHMI) along with Dr. Graham Hatfull and the SEA-PHAGES program for their support in our research.  

","authors":[{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology",""],"credit":["investigation","writing_originalDraft","writing_reviewEditing"],"email":"kmwprints@gmail.com","firstName":"Kit","lastName":"Williams","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":"0009-0000-3864-6059"},{"affiliations":["Southern Maine Community College, South Portland, ME, United States"],"departments":["Biology"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing"],"email":"esavage@mainecc.edu","firstName":"Emily","lastName":"Savage","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":"0009-0006-0316-2277"}],"awards":[],"conflictsOfInterest":"

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

","dataTable":{"url":null},"extendedData":[],"funding":"

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.

","image":{"url":"https://portal.micropublication.org/uploads/9b29c04ed3dccc206c62e0c4ead66bae.jpeg"},"imageCaption":"

Figure 1: A: Bacteriophage Wrackline forms clear plaques approximately 0.4 – 0.6mm in diameter (n=5). B: Transmission electron micrograph of bacteriophage Wrackline displaying a siphovirus morphology (Hitachi HT7800, 120kV, accelerating voltage 100kV) using negative stain (1% uranyl acetate). Wrackline has an icosahedral capsid 60nm in diameter and a tail of 150nm in length (n=1). Scale bar = 200 nm. C: Spot dilution assay of phage Wrackline and Cluster EE phage Gardevoir ( + control) plated with M. foliorum plated on PYCa, and Microbacterium sp. Casco Bay on Marine agar. Dilutions 10-5 through 10-7 show Wrackline shows similar plating efficiency on both bacterial hosts. 

","imageTitle":"

Plaques, TEM image, and host range assessment of Bacteriophage Wrackline

","methods":"

","reagents":"

Marine Agar Recipies for M. sp. Casco Bay

Marine Broth

750mL filtered seawater

250mL distilled water

0.5g yeast extract

0.5g casamino acids

3mL glycerine

Marine Agar

750mL filtered seawater

250mL distilled water

1g yeast extract

5g bacto peptone

15g agar

Marine Top Agar

750mL filtered seawater

250mL distilled water

0.5g yeast extract

0.5g bacto peptone

0.5g cas amino acids

5g agar

1mL of 1M CaCL2 per 100mL is added post-autoclave

PYCa Recipies for M. foliorum

PYCa Broth

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

PYCa Agar

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

15g agar

1mL cycloheximide (10mg/mL) added post-autoclave

PYCa Top Agar

1L distilled water

1g yeast extract

15g bacto peptone

2.5mL 40% dextrose

7g agar

1mL cycloheximide (10mg/mL) added post-autoclave

1mL of 1M CaCL2 per 100mL is added post-autoclave

","patternDescription":"

Gram-positive marine bacteria are understudied and typically challenging to culture (Gontang et al., 2007; Watkins 2022). Microbacterium sp. Casco Bay is halotolerant and simple to culture, making it an ideal host for the isolation and characterization of marine bacteriophage (Girard et al., 2025). While the whole genome of M. sp. Casco Bay has not yet been sequenced, 16S rRNA gene sequencing categorized it as a microbacterial strain. 

Bacteriophage Wrackline was isolated from a sand sample taken just above the high tide line on Willard Beach, South Portland, Maine (Global Positioning System [GPS] 43.645167 N, -70.227596 W). The phage was isolated using standard procedures (Zorawik et al., 2024).  Marine broth (Table 1) inoculated with M. sp. Casco Bay and five grams of sand were combined and incubated for 4 days at 30°C. The culture was filtered (0.22 μm pore size) and the filtrate spotted on to solidified marine top agar (Table 1) supplemented with M. sp. Casco Bay on a plate of marine agar (MA) (Table 1). After 48 hours at 30°C, Wrackline formed plaques with a clear center approximately 0.4–0.6mm wide (n=5) (Fig 1. A). Negative stain transmission electron microscopy showed siphovirus morphology with capsid size of 60nm and a 150nm long tail (n=1) (Fig 1. B). 

DNA was extracted from a Wrackline lysate using the Promega Wizard DNA clean-up kit. Sequencing library preparation was done using the NEB Ultra II Library Kit, and the library sequenced on the Illumina NextSeq 1000 sequencer with the XLEAP-PI kit. This yielded 2.6 million 100 base single-end reads, providing 5,885-fold coverage. Raw reads were trimmed and filtered by cutadapt 4.7 using the -nextseq-trim 30 argument (Martin 2011), and skewer 0.2.2 using options -q 20 -Q 30 -n -l 50 (Jiang et al., 2014) and genomic termini and completeness was checked using Consed V29 (Gordon and Green, 2013) default parameters. Wrackline's genome is 42,148 bp with a 69.2% GC content and is circularly permuted. 

Wrackline was automatically annotated in DNA Master v5.23.6, build 2705 (Pope and Jacobs-Sera, 2017) using Glimmer (v3.02b) (Delcher et al., 2007) and GeneMark (v2.5p) (Besemer and Borodovsky, 2005) to assess coding potential and identify putative genes. Start sites were then refined and gene functions predicted using Phamerator (Actino_Draft v597) (Cresawn et al., 2011), Starterator (v3.02) (Pacey, 2016), HHPred (v2.08 (PDB_mmCIF70, SCOPe70, Pfam-A, NCBI_Conserved_Domains [CD]) (Söding et al., 2005), and Blastp (Actinobacteriophage database, NCBI non-redundant database) (Altschul et al., 1990). DeepTMHMM (v1.2.33.c.) (Hallgren et al., 2022) and SOSUI (v1.11) (Hirokawa et al., 1998) were used to assess the presence of transmembrane proteins. No tRNA genes were detected using Aragorn (tRNA-scan SE v1.2.41 v). Default parameters were used for all software programs. 

This analysis identified 80 predicted protein-coding genes, 26 of which are orphams with no known homologs (Cresawn et al., 2011). The left arm of the genome is transcribed in the forward orientation and comprised of structural genes and the endolysin, followed by a reverse transcribed region containing genes of unknown functions and DNA binding domains, then returning to the forward orientation. The right arm of the genome contains a RecE-like exonuclease and RecT-like DNA pairing protein (GP 64 and 65) which may work together as a recombinase.  

Cluster assignment based on gene content similarity (GCS) of at least 35% to other phages in the Actinobacteria database (Actino_Draft database) placed Wrackline in cluster GF which only contains 3 other phages to date (Pope et al., 2017; Russell and Hatfull, 2017). The other cluster GF phages share more than 76.3% GCS, while Wrackline stands out sharing only 48.6% GCS with the group. While the structural genes of Wrackline are similar to other GF phages, the first 12 genes vary from others in the cluster and among those are 8 orphams for which no function was assigned. The reverse section of the genome from GP 38 – 60 shares some protein phams with other GFs, but is an orpham rich region, with 7 orphams with no functional assignment.  Additionally, other phages in the cluster were isolated using Microbacterium foliorum. Host range for phage Wrackline was evaluated by standard procedures (Zorawik et al., 2024). Spot plates were prepared using Wrackline and a control phage (Gardevoir (EE)) known to infect Microbacterium foliorum and Microbacterium sp. Casco Bay. Phage lysate for Wrackline and Gardevoir were diluted to 10-7 and 2uL were spotted onto each host. Wrackline is able to infect M. foliorum with a similar plating efficiency (Fig 1. C).  

Nucleotide sequence accession numbers

Wrackline is available at GenBank with Accession No. PV876983 and Sequence Read Archive (SRA) No. SRX29714291. The host strain Microbacterium sp. Casco Bay is archived in the National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Science (B81).

","references":[{"reference":"

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

Genome Sequence of Novel Cluster GF Bacteriophage Wrackline, Isolated on Microbacterium sp. Casco Bay

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