Corrigendum: Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda
Figgins V, et al. 2026. microPublication Biology. https://doi.org/10.17912/micropub.biology.001964
In the originally published article, co-author Savannah Prozik was inadvertently omitted from the author list. Her name now appears between Konur Onufer and Phoenix Smith.
","date":"Fri Apr 17 2026","correctionType":"corrigendum"}],"versions":[{"id":"b2da2bf3-1c95-42ad-843e-64b81ef24e51","decisionLetter":"Dear Margaret Saha,
Your article \"Sequence Analysis of two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda\" has been accepted, with minor modifications.
Review
Great description of two novel cluster B1 mycobacteriophages! I appreciated the wide variety of data offered in the figure and table. Below are my suggestions to clean up typos and improve clarity.
1. Figure: The plaque image scalebars report 12.5 nm instead of mm.
2. Figure legend: Sentence 1: Change to “Plaque morphologies of ElvisPhasley (a) and MesMerelda (b). ElvisPhasley forms clear plaques with translucent halos and Mesmerelda forms clear plaques.”
3. Figure legend: Dot plot. “Each line of the dot plot represents a phage” is misleading given that there are many lines of differing lengths. One could say that lines form between phage genome sequences with high nucleotide identity. I think it could also be more useful to label the X and Y access with where the B1 vs B2 genomes begin and end. If you know how to read a dot plot this is obvious but some of your readers may be new to dot plots. Given the high nucleotide identity shared across all of the B genomes, I am also not sure it is helpful to label the location of Mesmerelda and ElvisPhasely in the matrix. The labels as are of these two genomes doesn’t actually make sense given that the genomes run along the X and Y access and the lines are simple nucleotide identities between the sequences on the axes.
3. Figure legend: Dot plot. Typo. Change “gnomes” to genomes in sentence reading, “Both aces represent all 50 phage gnomes…”
4. Figure legend: genome maps. Change “Alignment of ElvisPhasely and Mesmerelda using Phamerator” to “Alignment of ElvisPhasely and Mesmerelda genomes using Phamerator.”
5. Paragraph 2: typo “Rsultant cultures” is misspelled.
6. Paragraph 2: 0.22-uM filter is used as an adjective and therefore there should be a hyphen between number and unit.
7. Paragraph 2: what strain of M. smegmatis used? MC2155?
8. Paragraph 2: “webbed plate” is slang. Instead: “flooding plates with nearly confluent bacterial lysis”
9. Table 1: Tail length: why is a ~ used to report average tail length? That feels unneeded when reporting standard error…. Also, is the standard error being reported or standard deviation? Perhaps label rows with particle dimensions as “Average tail length” and “Average capsid diameter.”
10. Paragraph 3: “Open reading frames (ORFs) were predicted using Glimmer…etc” Do you mean protein coding genes? There are lots of ORFs in the genomes but presumably we care about the ones that potentially code for proteins? Also Glimmer and GeneMark provide an auto-annotation. Did you not refine the annotation by verifying coding potential and then selecting translational starts that meet certain criteria? You mention examining start site similarity but it is not clear how that was used to manually determine starts and if other criteria (including all coding potential) were used to predict starts. Usually starts are chosen based on their inclusion of all predicted coding potential and conservation among homologs.
11. Paragraph 6: Sentence describing dot plot data refers to wrong panel in figure (reads 1f and should read 1e).
We kindly ask you to address each point and summarize your changes in line with the reviewer's response in the 'Comments to Editor' section on the platform. In order to expedite the processing of your revised manuscript, please be as specific as possible in your responses.
The microPublication Editorial Team
","decision":"revise","submitted":true,"abstract":"Mycobacteriophages ElvisPhasley and Mesmerelda were isolated from soil and infect Mycobacterium smegmatis. Each phage has siphovirus morphology and encodes 102 genes. Based on broader genomic similarity to actinobacteriophages, both are assigned to the B1 subcluster.
","acknowledgements":"We thank the University of Pittsburgh for genome sequencing, Old Dominion University Applied Research Center for the TEM images, the Hatfull lab, and the entire SEA-PHAGES program for their considerable ongoing support. We also thank the many institutions with SEA-PHAGES programs for their discovery and annotation of phages which made the Gepard plot in Figure 1f possible.
","authors":[{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"vafiggins@wm.edu","firstName":"Victoria","lastName":"Figgins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"gehussey@wm.edu","firstName":"Grace","lastName":"Hussey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"shaimowitz@wm.edu","firstName":"Sarah","lastName":"Haimowitz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"vpande@wm.edu","firstName":"Vera","lastName":"Pande","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"moroyster@wm.edu","firstName":"Marcus","lastName":"Royster","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egflanagan@wm.edu","firstName":"Emmery","lastName":"Flanagan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"mefountain@wm.edu","firstName":"Me'Shar'li'a","lastName":"Fountain","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egeorge@wm.edu","firstName":"Erin","lastName":"George","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zmherring@wm.edu","firstName":"Zion","lastName":"Herring","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"smjensen@wm.edu","firstName":"Scarlett","lastName":"Jensen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"jnnguyen@wm.edu","firstName":"Jenny","lastName":"Nguyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"klonufer@wm.edu","firstName":"Konur","lastName":"Onufer","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"plsmith02@wm.edu","firstName":"Phoenix","lastName":"Smith","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"bfstrong@wm.edu","firstName":"Brennan","lastName":"Strong","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zwang64@wm.edu","firstName":"Bruce","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"ayang02@wm.edu","firstName":"Abigail","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","validation","writing_reviewEditing","visualization"],"email":"hlqian@wm.edu","firstName":"Heather","lastName":"Qian","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"mssaha@wm.edu","firstName":"Margaret","lastName":"Saha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-0096-2667"}],"comments":"This manuscript is being submitted as part of the HHMI-microPublication workflow #HHMI_B4XYHB_2025_28.
","dataTable":null,"disclaimer":true,"funding":"","image":{"name":"Figure1.png","url":"https://portal.micropublication.org/uploads/86c99ad108dbcdb759e8377b03726a3e.png"},"imageCaption":"Plaque image of ElvisPhasley (a) showing clear plaques with translucent halos. and of Mesmerelda (b) showing clear plaques. Scale bars for plaque images are 12.5 mm. Negative stain (uranyl acetate, 1%) transmission electron microscopy (TEM) of ElvisPhasley (c) and Mesmerelda (d) revealing siphoviral morphology. Scale bar for micrographs are 50 nm. (e) Gepard plot comparing the genomic sequences of 40 B1 cluster phages, including ElvisPhasley and Mesmerelda, and 10 B2 cluster phages. Both axes represent all 50 phage gnomes, and each line of the dot plot represents a phage. The Gepard plot exemplifies the genomic similarities of ElvisPhasley and Mesmerelda to other B1 cluster phages and the genomic dissimilarity of ElvisPhasley and Mesmerelda to B2 cluster phages. (f) Alignment of ElvisPhasley and Mesmerelda using Phamerator. The genome is represented by the ruler, in kilo base pairs, with boxes above and below the ruler representing forward and reverse transcribed genes, respectively, and gene numbers presented within the box.
","imageTitle":"Plaque and TEM Images and Genomic Organization of ElvisPhasely and Mesmerelda
","laboratory":{"name":"","WBId":""},"methods":"","reagents":"","patternDescription":"Advancing the characterization of mycobacteriophages, bacteriophages that infect Mycobacterium hosts, is of interest due to increasing antibiotic resistance in pathogenic Mycobacterium species (Hatfull, 2020; Hatfull, 2022; Bonacorsi et al., 2024). Mycobacteriophage engineering is a promising approach in the detection and treatment of mycobacterial infections (Bonacorsi et al., 2024; Hosseiniporgham et al., 2022). Therefore, the continued characterization of mycobacteriophages broadens the capabilities of bioengineers in combating this global issue. Here, we report the genome sequences of two novel mycobacteriophages, ElvisPhasley and Mesmerelda.
Phages were isolated from two different soil samples: ElvisPhasley from wet, silty soil on the William & Mary campus in Williamsburg, VA; Mesmerelda from a bag of commercially available garden soil (Table 1). A standard enrichment procedure was followed for both samples: 5 g of each sample was suspended in 45 mL of 7H9 media, inoculated with Mycobacterium smegmatic mc2 155, and incubated in a 37°C shaker at 250 rpm for 48 hours (Zorawik et al., 2024). Rsultant cultures were filtered using a 0.22 µm filter, and filtrates were plated in 7H9 top agar with M. smegmatis. After 24-48 hours, both ElvisPhasley and Mesmerelda produced clear plaques (Fig. 1a-b). Both phages were purified with three rounds of plating, and a high titer lysate was prepared by flooding webbed plates with phage buffer (10 mM Tris, pH 7.5; 10 mM MgSO4; 68 mM NaCl; 1 mM CaCl2). Negative stain (1% uranyl acetate) transmission electron microscopy of each lysate revealed siphovirus morphology for both phages (Fig. 1c-d).
Phage DNA was extracted from each high titer lysate using a phenol-chloroform-isoamyl alcohol procedure and ethanol precipitation (Sambrook and Russell, 2006). DNA was prepared for sequencing using the NEB Ultra II Library Kit and sequenced using an Illumina NextSeq 1000 sequencer (single-end, 100 base read). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Newbler (v.2.9) was then used to assemble the genome and Consed (v.29) to check for completeness (Russell, 2018; Gordon et al., 1998). Sequencing data and genome characteristics are presented in Table 1.
Genome annotation was performed using DNA Master (v.5.23.6) and PECAAN (v.20221109) (Rinehart et al., 2016; Pope and Jacobs-Sera, 2018). Open reading frames (ORFs) were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007), coding potential was verified using GeneMark (Besemer and Borodovsky, 2005), and start site similarity in homologs was found using BLASTp and Starterator (http://phages.wustl.edu/starterator/) against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990). No tRNAs were predicted using Aragorn (v.1.2.41) and tRNAscan (Laslett and Canback, 2004; Lowe and Eddy, 1997).
Predictions from HHPred (using the PDB_mmCIF70, NCBI_CD, SCOPe70, and pFAM-A databases), BLASTp, and Phamerator for highly similar genes were used to assign putative gene functions (Cresawn et al., 2011; Zimmermann et al., 2018). Both phages are assigned to cluster B, subcluster B1 using the gene content similarity (GCS) tool at the Actinobacteriophage database, PhagesDB, and clustering parameters of at least 35% GCS to actinobacteriophages (Russell and Hatfull, 2017). Default settings were used for all software.
The genomes of ElvisPhasley and Mesmerelda both encode 102 protein-coding genes, with 33 and 32 genes, respectively, assigned functions involved in virion propagation, structure, and lysis. As neither ElvisPhasley nor Mesmerelda encode a putative integrase or other proteins implicated in lysogeny, these phages are predicted to be virulent. Both ElvisPhasley and Mesmerelda display strong genomic similarity to other B1 subcluster phages and genomic dissimilarity to B2 subcluster phages as displayed on a Gepard dot plot (Fig. 1f; Krumsiek et al., 2007). ElvisPhasley shares 89.22% GCS with its closest relative, OSMaximus (Russell and Hatfull, 2017). At the nucleotide level, ElvisPhasley shares 99.00% identity with OSMaximus over 97% coverage (BLAST). Within this covered region, we identified 968 nucleotide differences, 249 of which were found in coding regions. Of these differences, 238 resulted in amino acid substitutions, 98 of which were conservative and 140 of which were non-conservative as classified by the BLOSUM62 alignment score matrix. Likewise, Mesmerelda shares 95.1% GCS with its closest relative, Orfeu (Russell and Hatfull, 2017). These phages share 99.16% nucleotide identity over 100% coverage. Of the 557 nucleotide differences, 136 differences occur in coding regions. These differences result in 115 amino acid substitutions, 53 of which are conservative and 62 of which are not conservative.
Nucleotide sequence accession numbers
ElvisPhasley and Mesmerelda are available at GenBank with Accession No. PV876949 and PV876954, and Sequence Read Archive (SRA) No. SRX29990110 and SRX29990101.
Table 1: Genome and sequencing information for Mesmerelda and ElvisPhasley
Phage | ElvisPhasley | Mesmerelda |
Isolation GPS Coordinates | 37° 16' 13.4034\" N 76° 43' 3.216\" W | 37° 16' 11.8992\" N 76° 42' 52.5996\" W |
Morphology | Siphovirus | Siphovirus |
Capsid diameter | 60 ± 4 nm (n = 5) | 50 ± 4 nm (n = 5) |
Tail length | ~310 ± 10 nm (n = 5) | ~220 ± 10 nm (n = 5) |
Sequencing reads | 4,697,092 | 4,305,175 |
Sequencing coverage, fold | 6,513 | 5,969 |
Genome length (bp) | 69,502 | 68,890 |
Character of genome ends | Circularly permuted | Circularly permuted |
Number of protein-coding genes | 102 | 102 |
GC content (%) | 66.4 | 66.4 |
Accession number | PV876949 | PV876954 |
SRA | SRX29990110 | SRX29990101 |
Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Bonacorsi A, Ferretti C, Di Luca M, Rindi L. 2024. Mycobacteriophages and Their Applications. Antibiotics. 13: 926.","pubmedId":"","doi":"10.3390/antibiotics13100926"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. Cresawn2011.","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.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, dynamics, and applications. Annu. Rev. Virol. 7: 37-61.","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLoS Pathog. 18: e1010602.","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Hosseiniporgham S, Sechi LA. 2022. A Review on Mycobacteriophages: From Classification to Applications. Pathogens. 11: 777.","pubmedId":"","doi":"10.3390/pathogens11070777"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. Jiang2014.","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Krumsiek J, Arnold R, Rattei T. 2007. Gepard: a rapid and sensitive tool for creating dotplots on genome scale.
","pubmedId":"","doi":"10.1093/bioinformatics/btm039"},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide. Nucleic Acids Res. 32: 11-16.","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17: 10.","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Pope WH, Jacobs Sera D. 2018. Annotation of bacteriophage genome sequences using DNA Master: An overview. Methods Mol. Biol. 1681: 217-229.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Rinehart CA, Gaffney B, Wood JD, Smith S. 2016. PECAAN: Phage Evidence Collection And Annotation Network.","pubmedId":"","doi":""},{"reference":"Russell DA. 2018. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Russell2018.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Sambrook J, Russell DW. 2006. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006: db.prot4455.","pubmedId":"","doi":"10.1101/pdb.prot4455"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long. PLoS Comput. Biol. 13: e1005595.","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, et al., Alva V. 2018. undefined. Computation Resources for Molecular Biology. 430: 2237.","pubmedId":"","doi":"https://doi.org/10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs Sera D, Freise AC, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Zorawik2024.","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"suggestedReviewer":{"name":"N/A
","WBId":""},"title":"Sequence Analysis of two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda
","reviews":[{"reviewer":{"displayName":"Sally Molloy"},"openAcknowledgement":false,"status":{"submitted":true}},{"reviewer":{"displayName":"Dan Williams"},"openAcknowledgement":false,"status":{"submitted":true}}]},{"id":"05c267cb-b620-4617-a444-62267476f716","decisionLetter":"Dear Margaret Saha,
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","decision":"accept","submitted":true,"abstract":"Mycobacteriophages ElvisPhasley and Mesmerelda were isolated from soil and infect Mycobacterium smegmatis. Each phage has siphovirus morphology and encodes 102 genes. Based on broader genomic similarity to actinobacteriophages, both are assigned to the B1 subcluster.
","acknowledgements":"We thank the University of Pittsburgh for genome sequencing, Old Dominion University Applied Research Center for the TEM images, the Hatfull lab, and the entire SEA-PHAGES program for their considerable ongoing support. We also thank the many institutions with SEA-PHAGES programs for their discovery and annotation of phages which made the Gepard plot in Figure 1f possible.
","authors":[{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"vafiggins@wm.edu","firstName":"Victoria","lastName":"Figgins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"gehussey@wm.edu","firstName":"Grace","lastName":"Hussey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"shaimowitz@wm.edu","firstName":"Sarah","lastName":"Haimowitz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"vpande@wm.edu","firstName":"Vera","lastName":"Pande","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"moroyster@wm.edu","firstName":"Marcus","lastName":"Royster","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egflanagan@wm.edu","firstName":"Emmery","lastName":"Flanagan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"mefountain@wm.edu","firstName":"Me'Shar'li'a","lastName":"Fountain","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egeorge@wm.edu","firstName":"Erin","lastName":"George","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zmherring@wm.edu","firstName":"Zion","lastName":"Herring","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"smjensen@wm.edu","firstName":"Scarlett","lastName":"Jensen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"jnnguyen@wm.edu","firstName":"Jenny","lastName":"Nguyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"klonufer@wm.edu","firstName":"Konur","lastName":"Onufer","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"plsmith02@wm.edu","firstName":"Phoenix","lastName":"Smith","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"bfstrong@wm.edu","firstName":"Brennan","lastName":"Strong","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zwang64@wm.edu","firstName":"Bruce","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"ayang02@wm.edu","firstName":"Abigail","lastName":"Yang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["formalAnalysis","validation","writing_reviewEditing","visualization"],"email":"hlqian@wm.edu","firstName":"Heather","lastName":"Qian","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"mssaha@wm.edu","firstName":"Margaret","lastName":"Saha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-0096-2667"}],"comments":"Dear Editor, Thank you for your comments. We agree with every one of them and have made all the requested edits/changes as described below.
1. Figure: The plaque image scalebars report 12.5 nm instead of mm.
>>>Response: We apologize for this oversight, and we have now updated the scale bar in Fig. 1a and 1b to read “12.5 mm” instead of “12.5 nm”.
2. Figure legend: Sentence 1: Change to “Plaque morphologies of ElvisPhasley (a) and MesMerelda (b). ElvisPhasley forms clear plaques with translucent halos and Mesmerelda forms clear plaques.”
>>>Response: We agree that this rephrasing of the first sentence in the figure legend improves clarity. We have now revised the opening sentence of the figure legend based on the suggested changes to read: “Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques.”
3a. Figure legend: Dot plot. “Each line of the dot plot represents a phage” is misleading given that there are many lines of differing lengths. One could say that lines form between phage genome sequences with high nucleotide identity. I think it could also be more useful to label the X and Y access with where the B1 vs B2 genomes begin and end. If you know how to read a dot plot this is obvious but some of your readers may be new to dot plots. Given the high nucleotide identity shared across all of the B genomes, I am also not sure it is helpful to label the location of Mesmerelda and ElvisPhasely in the matrix. The labels as are of these two genomes doesn’t actually make sense given that the genomes run along the X and Y access and the lines are simple nucleotide identities between the sequences on the axes.
>>>Response: We agree that our description of the Gepard dot plot was misleading and have now updated the figure legend to read: “Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences.” Additionally, we have removed the labels for ElvisPhasley and Mesmerelda on the Gepard plot because we agree that these labels are unnecessary. Finally, labels have been added to the X and Y axes of the Gepard plot to indicate which dots refer to B1 phages and which refer to B2 phages.
3b. Figure legend: Dot plot. Typo. Change “gnomes” to genomes in sentence reading, “Both aces represent all 50 phage gnomes…”
>>>Response: We apologize for missing this typo, and we have now updated the manuscript to read: “Both axes represent all 50 phage genomes…”.
4. Figure legend: genome maps. Change “Alignment of ElvisPhasely and Mesmerelda using Phamerator” to “Alignment of ElvisPhasely and Mesmerelda genomes using Phamerator.”
>>>Response: We agree that adding the word “genomes” to “Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator” in the figure legend improves clarity, and we have updated the figure legend to reflect this suggestion.
5. Paragraph 2: typo “Rsultant cultures” is misspelled.
>>>Response: Once again, we apologize for this typo and have updated Paragraph 2 of the manuscript to read: “Resultant cultures were filtered…”.
6. Paragraph 2: 0.22-uM filter is used as an adjective and therefore there should be a hyphen between number and unit.
>>>Response: We appreciate this correction, and we have now updated the text of Paragraph 2 to read: “…filtered using a 0.22-µm filter…” with the suggested hyphen between the unit and number.
7. Paragraph 2: what strain of M. smegmatis used? MC2155?
>>>Response: We have now updated both sentences referencing M. smegmatis to specify that the M. smegmatis mc2 155 strain was used instead of just the first sentence.
8. Paragraph 2: “webbed plate” is slang. Instead: “flooding plates with nearly confluent bacterial lysis”
>>>Response: We apologize for our use of slang in the manuscript, and we have updated the text in Paragraph 2 to read: “…a high titer lysate was prepared by flooding plates that exhibited nearly confluent bacterial lysis with phage buffer” per the suggested rewording.
9. Table 1: Tail length: why is a ~ used to report average tail length? That feels unneeded when reporting standard error…. Also, is the standard error being reported or standard deviation? Perhaps label rows with particle dimensions as “Average tail length” and “Average capsid diameter.”
>>>Response: We agree that including the “~” symbol in table to report average tail length is unnecessary and have therefore removed it. Additionally, we have accepted the suggestion to label the rows on Table 1 with particle dimensions as “Average tail length” and “Average capsid diameter”. Finally, we would like to confirm that we report the standard deviation of average tail length and capsid diameter, not standard error. To address this in our table, we have added (± SD) to “Average capsid diameter (± SD)” and “Average tail length (± SD)” for additional clarity.
10. Paragraph 3: “Open reading frames (ORFs) were predicted using Glimmer…etc” Do you mean protein coding genes? There are lots of ORFs in the genomes but presumably we care about the ones that potentially code for proteins? Also Glimmer and GeneMark provide an auto-annotation. Did you not refine the annotation by verifying coding potential and then selecting translational starts that meet certain criteria? You mention examining start site similarity but it is not clear how that was used to manually determine starts and if other criteria (including all coding potential) were used to predict starts. Usually starts are chosen based on their inclusion of all predicted coding potential and conservation among homologs.
>>>Response: We agree that revising the text of Paragraph 4 to read “Protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5)…” is more accurate than “Open reading frames (ORFs) were predicted…” and have updated the manuscript to reflect this change. Additionally, to provide greater clarity on how we determined the start site of each gene, we have updated the text of Paragraph 4 to read, “Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007. These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990).”
11. Paragraph 6: Sentence describing dot plot data refers to wrong panel in figure (reads 1f and should read 1e).
>>>Response: We apologize for overlooking this incorrect reference to Fig 1f instead of Fig 1e. We have relabeled the sub-figures so that the Phamerator map is now Fig 1e and the Gepard plot is Fig 1f to reflect the order in which each sub-figure is referenced in the manuscript. As such, the reference to the Gepard plot in the manuscript still reads “(Fig. 1f)”, but this is now consistent with the labeling of Figure 1 and its figure legend.
We thank the reviewer and editor for their helpful comments.
On behalf of the authors,
Margaret Saha
Professor of Applied Science
This manuscript is being submitted as part of the HHMI-microPublication workflow #HHMI_B4XYHB_2025_28.
","dataTable":null,"disclaimer":true,"funding":"","image":{"name":"260308_2018_ManuscriptFigure.jpg","url":"https://portal.micropublication.org/uploads/8326d1e514704e97e897c4c193440fb1.jpg"},"imageCaption":"Fig. 1 Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques. Scale bars for plaque images are 12.5 mm. Negative stain (uranyl acetate, 1%) transmission electron microscopy (TEM) of ElvisPhasley (c) and Mesmerelda (d) revealing siphoviral morphology. Scale bars for micrographs are 50 nm. (e) Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator. The genome is represented by the ruler, in kilo base pairs, with boxes above and below the ruler representing forward and reverse transcribed genes, respectively, and gene numbers presented within the box. (f) Gepard plot comparing the genomic sequences of 40 B1 cluster phages, including ElvisPhasley and Mesmerelda, and 10 B2 cluster phages. Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences. The Gepard plot exemplifies the genomic similarities of ElvisPhasley and Mesmerelda to other B1 cluster phages and the genomic dissimilarity of ElvisPhasley and Mesmerelda to B2 cluster phages.
","imageTitle":"Plaque and TEM Images and Genomic Organization of ElvisPhasely and Mesmerelda
","laboratory":{"name":"","WBId":""},"methods":"","reagents":"","patternDescription":"Advancing the characterization of mycobacteriophages, bacteriophages that infect Mycobacterium hosts, is of interest due to increasing antibiotic resistance in pathogenic Mycobacterium species (Hatfull, 2020; Hatfull, 2022; Bonacorsi et al., 2024). Mycobacteriophage engineering is a promising approach in the detection and treatment of mycobacterial infections (Bonacorsi et al., 2024; Hosseiniporgham et al., 2022). Therefore, the continued characterization of mycobacteriophages broadens the capabilities of bioengineers in combating this global issue. Here, we report the genome sequences of two novel mycobacteriophages, ElvisPhasley and Mesmerelda.
Phages were isolated from two different soil samples: ElvisPhasley from wet, silty soil on the William & Mary campus in Williamsburg, VA; Mesmerelda from a bag of commercially available garden soil (Table 1). A standard enrichment procedure was followed for both samples: 5 g of each sample was suspended in 45 mL of 7H9 media, inoculated with Mycobacterium smegmatis mc2 155, and incubated in a 37°C shaker at 250 rpm for 48 hours (Zorawik et al., 2024). Resultant cultures were filtered using a 0.22-µm filter, and filtrates were plated in 7H9 top agar with M. smegmatis mc2 155. After 24-48 hours, both ElvisPhasley and Mesmerelda produced clear plaques (Fig. 1a-b). Both phages were purified with three rounds of plating, and a high titer lysate was prepared by flooding plates exhibiting nearly confluent bacterial lysis with phage buffer (10 mM Tris, pH 7.5; 10 mM MgSO4; 68 mM NaCl; 1 mM CaCl2). Negative stain (1% uranyl acetate) transmission electron microscopy of each lysate revealed siphovirus morphology for both phages (Fig. 1c-d).
Phage DNA was extracted from each high titer lysate using a phenol-chloroform-isoamyl alcohol procedure and ethanol precipitation (Sambrook and Russell, 2006). DNA was prepared for sequencing using the NEB Ultra II Library Kit and sequenced using an Illumina NextSeq 1000 sequencer (single-end, 100 base read). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Newbler (v.2.9) was then used to assemble the genome and Consed (v.29) to check for completeness (Russell, 2018; Gordon et al., 1998). Sequencing data and genome characteristics are presented in Table 1.
Genome annotation was performed using DNA Master (v.5.23.6) and PECAAN (v.20221109) (Rinehart et al., 2016; Pope and Jacobs-Sera, 2018). Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007. These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990). No tRNAs were predicted using Aragorn (v.1.2.41) and tRNAscan (Laslett and Canback, 2004; Lowe and Eddy, 1997).
Predictions from HHPred (using the PDB_mmCIF70, NCBI_CD, SCOPe70, and pFAM-A databases), BLASTp, and Phamerator for highly similar genes were used to assign putative gene functions (Cresawn et al., 2011; Zimmermann et al., 2018). Both phages are assigned to cluster B, subcluster B1 using the gene content similarity (GCS) tool at the Actinobacteriophage database, PhagesDB, and clustering parameters of at least 35% GCS to actinobacteriophages (Russell and Hatfull, 2017). Default settings were used for all software.
The genomes of ElvisPhasley and Mesmerelda both encode 102 protein-coding genes, with 33 and 32 genes, respectively, assigned functions involved in virion propagation, structure, and lysis (Fig. 1e). As neither ElvisPhasley nor Mesmerelda encode a putative integrase or other proteins implicated in lysogeny, these phages are predicted to be virulent. Both ElvisPhasley and Mesmerelda display strong genomic similarity to other B1 subcluster phages and genomic dissimilarity to B2 subcluster phages as displayed on a Gepard dot plot (Fig. 1f; Krumsiek et al., 2007). ElvisPhasley shares 89.22% GCS with its closest relative, OSMaximus (Russell and Hatfull, 2017). At the nucleotide level, ElvisPhasley shares 99.00% identity with OSMaximus over 97% coverage (BLAST). Within this covered region, we identified 968 nucleotide differences, 249 of which were found in coding regions. Of these differences, 238 resulted in amino acid substitutions, 98 of which were conservative and 140 of which were non-conservative as classified by the BLOSUM62 alignment score matrix. Likewise, Mesmerelda shares 95.1% GCS with its closest relative, Orfeu (Russell and Hatfull, 2017). These phages share 99.16% nucleotide identity over 100% coverage. Of the 557 nucleotide differences, 136 differences occur in coding regions. These differences result in 115 amino acid substitutions, 53 of which are conservative and 62 of which are not conservative.
Nucleotide sequence accession numbers
ElvisPhasley and Mesmerelda are available at GenBank with Accession No. PV876949 and PV876954, and Sequence Read Archive (SRA) No. SRX29990110 and SRX29990101.
Table 1: Genome and sequencing information for Mesmerelda and ElvisPhasley
Phage | ElvisPhasley | Mesmerelda |
Isolation GPS coordinates | 37° 16' 13.4034\" N 76° 43' 3.216\" W | 37° 16' 11.8992\" N 76° 42' 52.5996\" W |
Morphology | Siphovirus | Siphovirus |
Average capsid diameter (± SD) | 60 ± 4 nm (n = 5) | 50 ± 4 nm (n = 5) |
Average tail length (± SD) | 310 ± 10 nm (n = 5) | 220 ± 10 nm (n = 5) |
Sequencing reads | 4,697,092 | 4,305,175 |
Sequencing coverage, fold | 6,513 | 5,969 |
Genome length (bp) | 69,502 | 68,890 |
Character of genome ends | Circularly permuted | Circularly permuted |
Number of protein-coding genes | 102 | 102 |
GC content (%) | 66.4 | 66.4 |
Accession number | ||
SRA |
Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Bonacorsi A, Ferretti C, Di Luca M, Rindi L. 2024. Mycobacteriophages and Their Applications. Antibiotics. 13: 926.","pubmedId":"","doi":"10.3390/antibiotics13100926"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. Cresawn2011.","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.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, dynamics, and applications. Annu. Rev. Virol. 7: 37-61.","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLoS Pathog. 18: e1010602.","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Hosseiniporgham S, Sechi LA. 2022. A Review on Mycobacteriophages: From Classification to Applications. Pathogens. 11: 777.","pubmedId":"","doi":"10.3390/pathogens11070777"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. Jiang2014.","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Krumsiek J, Arnold R, Rattei T. 2007. Gepard: a rapid and sensitive tool for creating dotplots on genome scale.
","pubmedId":"","doi":"10.1093/bioinformatics/btm039"},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide. Nucleic Acids Res. 32: 11-16.","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17: 10.","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Pope WH, Jacobs Sera D. 2018. Annotation of bacteriophage genome sequences using DNA Master: An overview. Methods Mol. Biol. 1681: 217-229.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Rinehart CA, Gaffney B, Wood JD, Smith S. 2016. PECAAN: Phage Evidence Collection And Annotation Network.","pubmedId":"","doi":""},{"reference":"Russell DA. 2018. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Russell2018.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Sambrook J, Russell DW. 2006. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006: db.prot4455.","pubmedId":"","doi":"10.1101/pdb.prot4455"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long. PLoS Comput. Biol. 13: e1005595.","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, et al., Alva V. 2018. undefined. Computation Resources for Molecular Biology. 430: 2237.","pubmedId":"","doi":"https://doi.org/10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs Sera D, Freise AC, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Zorawik2024.","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"suggestedReviewer":{"name":"N/A
","WBId":""},"title":"Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda
","reviews":[]},{"id":"00b4cdc8-153f-4811-99d1-d7fc66c13107","decisionLetter":null,"decision":"edit","submitted":true,"abstract":"Mycobacteriophages ElvisPhasley and Mesmerelda were isolated from soil and infect Mycobacterium smegmatis. Each phage has siphovirus morphology and encodes 102 genes. Based on broader genomic similarity to actinobacteriophages, both are assigned to the B1 subcluster.
","acknowledgements":"We thank the University of Pittsburgh for genome sequencing, Old Dominion University Applied Research Center for the TEM images, the Hatfull lab, and the entire SEA-PHAGES program for their considerable ongoing support. We also thank the many institutions with SEA-PHAGES programs for their discovery and annotation of phages which made the Gepard plot in Figure 1f possible.
","authors":[{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"vafiggins@wm.edu","firstName":"Victoria","lastName":"Figgins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"gehussey@wm.edu","firstName":"Grace","lastName":"Hussey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"shaimowitz@wm.edu","firstName":"Sarah","lastName":"Haimowitz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"vpande@wm.edu","firstName":"Vera","lastName":"Pande","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"moroyster@wm.edu","firstName":"Marcus","lastName":"Royster","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egflanagan@wm.edu","firstName":"Emmery","lastName":"Flanagan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"mefountain@wm.edu","firstName":"Me'Shar'li'a","lastName":"Fountain","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egeorge@wm.edu","firstName":"Erin","lastName":"George","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zmherring@wm.edu","firstName":"Zion","lastName":"Herring","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"smjensen@wm.edu","firstName":"Scarlett","lastName":"Jensen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"jnnguyen@wm.edu","firstName":"Jenny","lastName":"Nguyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"klonufer@wm.edu","firstName":"Konur","lastName":"Onufer","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"plsmith02@wm.edu","firstName":"Phoenix","lastName":"Smith","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"bfstrong@wm.edu","firstName":"Brennan","lastName":"Strong","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zwang64@wm.edu","firstName":"Bruce","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"ayang02@wm.edu","firstName":"Abigail","lastName":"Yang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["formalAnalysis","validation","writing_reviewEditing","visualization"],"email":"hlqian@wm.edu","firstName":"Heather","lastName":"Qian","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William and Mary, Williamsburg, VA USA"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"mssaha@wm.edu","firstName":"Margaret","lastName":"Saha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-0096-2667"}],"comments":"Dear Editor, Thank you for your comments. We agree with every one of them and have made all the requested edits/changes as described below.
1. Figure: The plaque image scalebars report 12.5 nm instead of mm.
>>>Response: We apologize for this oversight, and we have now updated the scale bar in Fig. 1a and 1b to read “12.5 mm” instead of “12.5 nm”.
2. Figure legend: Sentence 1: Change to “Plaque morphologies of ElvisPhasley (a) and MesMerelda (b). ElvisPhasley forms clear plaques with translucent halos and Mesmerelda forms clear plaques.”
>>>Response: We agree that this rephrasing of the first sentence in the figure legend improves clarity. We have now revised the opening sentence of the figure legend based on the suggested changes to read: “Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques.”
3a. Figure legend: Dot plot. “Each line of the dot plot represents a phage” is misleading given that there are many lines of differing lengths. One could say that lines form between phage genome sequences with high nucleotide identity. I think it could also be more useful to label the X and Y access with where the B1 vs B2 genomes begin and end. If you know how to read a dot plot this is obvious but some of your readers may be new to dot plots. Given the high nucleotide identity shared across all of the B genomes, I am also not sure it is helpful to label the location of Mesmerelda and ElvisPhasely in the matrix. The labels as are of these two genomes doesn’t actually make sense given that the genomes run along the X and Y access and the lines are simple nucleotide identities between the sequences on the axes.
>>>Response: We agree that our description of the Gepard dot plot was misleading and have now updated the figure legend to read: “Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences.” Additionally, we have removed the labels for ElvisPhasley and Mesmerelda on the Gepard plot because we agree that these labels are unnecessary. Finally, labels have been added to the X and Y axes of the Gepard plot to indicate which dots refer to B1 phages and which refer to B2 phages.
3b. Figure legend: Dot plot. Typo. Change “gnomes” to genomes in sentence reading, “Both aces represent all 50 phage gnomes…”
>>>Response: We apologize for missing this typo, and we have now updated the manuscript to read: “Both axes represent all 50 phage genomes…”.
4. Figure legend: genome maps. Change “Alignment of ElvisPhasely and Mesmerelda using Phamerator” to “Alignment of ElvisPhasely and Mesmerelda genomes using Phamerator.”
>>>Response: We agree that adding the word “genomes” to “Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator” in the figure legend improves clarity, and we have updated the figure legend to reflect this suggestion.
5. Paragraph 2: typo “Rsultant cultures” is misspelled.
>>>Response: Once again, we apologize for this typo and have updated Paragraph 2 of the manuscript to read: “Resultant cultures were filtered…”.
6. Paragraph 2: 0.22-uM filter is used as an adjective and therefore there should be a hyphen between number and unit.
>>>Response: We appreciate this correction, and we have now updated the text of Paragraph 2 to read: “…filtered using a 0.22-µm filter…” with the suggested hyphen between the unit and number.
7. Paragraph 2: what strain of M. smegmatis used? MC2155?
>>>Response: We have now updated both sentences referencing M. smegmatis to specify that the M. smegmatis mc2 155 strain was used instead of just the first sentence.
8. Paragraph 2: “webbed plate” is slang. Instead: “flooding plates with nearly confluent bacterial lysis”
>>>Response: We apologize for our use of slang in the manuscript, and we have updated the text in Paragraph 2 to read: “…a high titer lysate was prepared by flooding plates that exhibited nearly confluent bacterial lysis with phage buffer” per the suggested rewording.
9. Table 1: Tail length: why is a ~ used to report average tail length? That feels unneeded when reporting standard error…. Also, is the standard error being reported or standard deviation? Perhaps label rows with particle dimensions as “Average tail length” and “Average capsid diameter.”
>>>Response: We agree that including the “~” symbol in table to report average tail length is unnecessary and have therefore removed it. Additionally, we have accepted the suggestion to label the rows on Table 1 with particle dimensions as “Average tail length” and “Average capsid diameter”. Finally, we would like to confirm that we report the standard deviation of average tail length and capsid diameter, not standard error. To address this in our table, we have added (± SD) to “Average capsid diameter (± SD)” and “Average tail length (± SD)” for additional clarity.
10. Paragraph 3: “Open reading frames (ORFs) were predicted using Glimmer…etc” Do you mean protein coding genes? There are lots of ORFs in the genomes but presumably we care about the ones that potentially code for proteins? Also Glimmer and GeneMark provide an auto-annotation. Did you not refine the annotation by verifying coding potential and then selecting translational starts that meet certain criteria? You mention examining start site similarity but it is not clear how that was used to manually determine starts and if other criteria (including all coding potential) were used to predict starts. Usually starts are chosen based on their inclusion of all predicted coding potential and conservation among homologs.
>>>Response: We agree that revising the text of Paragraph 4 to read “Protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5)…” is more accurate than “Open reading frames (ORFs) were predicted…” and have updated the manuscript to reflect this change. Additionally, to provide greater clarity on how we determined the start site of each gene, we have updated the text of Paragraph 4 to read, “Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007. These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990).”
11. Paragraph 6: Sentence describing dot plot data refers to wrong panel in figure (reads 1f and should read 1e).
>>>Response: We apologize for overlooking this incorrect reference to Fig 1f instead of Fig 1e. We have relabeled the sub-figures so that the Phamerator map is now Fig 1e and the Gepard plot is Fig 1f to reflect the order in which each sub-figure is referenced in the manuscript. As such, the reference to the Gepard plot in the manuscript still reads “(Fig. 1f)”, but this is now consistent with the labeling of Figure 1 and its figure legend.
We thank the reviewer and editor for their helpful comments.
On behalf of the authors,
Margaret Saha
Professor of Applied Science
This manuscript is being submitted as part of the HHMI-microPublication workflow #HHMI_B4XYHB_2025_28.
","dataTable":null,"disclaimer":true,"funding":"","image":{"name":"260308_2018_ManuscriptFigure.jpg","url":"https://portal.micropublication.org/uploads/8326d1e514704e97e897c4c193440fb1.jpg"},"imageCaption":"Fig. 1 Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques. Scale bars for plaque images are 12.5 mm. Negative stain (uranyl acetate, 1%) transmission electron microscopy (TEM) of ElvisPhasley (c) and Mesmerelda (d) revealing siphoviral morphology. Scale bars for micrographs are 50 nm. (e) Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator. The genome is represented by the ruler, in kilo base pairs, with boxes above and below the ruler representing forward and reverse transcribed genes, respectively, and gene numbers presented within the box. (f) Gepard plot comparing the genomic sequences of 40 B1 cluster phages, including ElvisPhasley and Mesmerelda, and 10 B2 cluster phages. Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences. The Gepard plot exemplifies the genomic similarities of ElvisPhasley and Mesmerelda to other B1 cluster phages and the genomic dissimilarity of ElvisPhasley and Mesmerelda to B2 cluster phages.
","imageTitle":"Plaque and TEM Images and Genomic Organization of ElvisPhasley and Mesmerelda
","laboratory":{"name":"","WBId":""},"methods":"","reagents":"","patternDescription":"Advancing the characterization of mycobacteriophages, bacteriophages that infect Mycobacterium hosts, is of interest due to increasing antibiotic resistance in pathogenic Mycobacterium species (Hatfull, 2020; Hatfull, 2022; Bonacorsi et al., 2024). Mycobacteriophage engineering is a promising approach in the detection and treatment of mycobacterial infections (Bonacorsi et al., 2024; Hosseiniporgham et al., 2022). Therefore, the continued characterization of mycobacteriophages broadens the capabilities of bioengineers in combating this global issue. Here, we report the genome sequences of two novel mycobacteriophages, ElvisPhasley and Mesmerelda.
Phages were isolated from two different soil samples: ElvisPhasley from wet, silty soil on the William & Mary campus in Williamsburg, VA; Mesmerelda from a bag of commercially available garden soil (Table 1). A standard enrichment procedure was followed for both samples: 5 g of each sample was suspended in 45 mL of 7H9 media, inoculated with Mycobacterium smegmatis mc2 155, and incubated in a 37°C shaker at 250 rpm for 48 hours (Zorawik et al., 2024). Resultant cultures were filtered using a 0.22-µm filter, and filtrates were plated in 7H9 top agar with M. smegmatis mc2 155. After 24-48 hours, both ElvisPhasley and Mesmerelda produced clear plaques (Fig. 1a-b). Both phages were purified with three rounds of plating, and a high titer lysate was prepared by flooding plates exhibiting nearly confluent bacterial lysis with phage buffer (10 mM Tris, pH 7.5; 10 mM MgSO4; 68 mM NaCl; 1 mM CaCl2). Negative stain (1% uranyl acetate) transmission electron microscopy of each lysate revealed siphovirus morphology for both phages (Fig. 1c-d).
Phage DNA was extracted from each high titer lysate using a phenol-chloroform-isoamyl alcohol procedure and ethanol precipitation (Sambrook and Russell, 2006). DNA was prepared for sequencing using the NEB Ultra II Library Kit and sequenced using an Illumina NextSeq 1000 sequencer (single-end, 100 base read). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Newbler (v.2.9) was then used to assemble the genome and Consed (v.29) to check for completeness (Russell, 2018; Gordon et al., 1998). Sequencing data and genome characteristics are presented in Table 1.
Genome annotation was performed using DNA Master (v.5.23.6) and PECAAN (v.20221109) (Rinehart et al., 2016; Pope and Jacobs-Sera, 2018). Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007). These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990). No tRNAs were predicted using Aragorn (v.1.2.41) and tRNAscan (Laslett and Canback, 2004; Lowe and Eddy, 1997).
Predictions from HHPred (using the PDB_mmCIF70, NCBI_CD, SCOPe70, and pFAM-A databases), BLASTp, and Phamerator for highly similar genes were used to assign putative gene functions (Cresawn et al., 2011; Zimmermann et al., 2018). Both phages are assigned to cluster B, subcluster B1 using the gene content similarity (GCS) tool at the Actinobacteriophage database, PhagesDB, and clustering parameters of at least 35% GCS to actinobacteriophages (Russell and Hatfull, 2017). Default settings were used for all software.
The genomes of ElvisPhasley and Mesmerelda both encode 102 protein-coding genes, with 33 and 32 genes, respectively, assigned functions involved in virion propagation, structure, and lysis (Fig. 1e). As neither ElvisPhasley nor Mesmerelda encode a putative integrase or other proteins implicated in lysogeny, these phages are predicted to be virulent. Both ElvisPhasley and Mesmerelda display strong genomic similarity to other B1 subcluster phages and genomic dissimilarity to B2 subcluster phages as displayed on a Gepard dot plot (Fig. 1f; Krumsiek et al., 2007). ElvisPhasley shares 89.22% GCS with its closest relative, OSMaximus (Russell and Hatfull, 2017). At the nucleotide level, ElvisPhasley shares 99.00% identity with OSMaximus over 97% coverage (BLAST). Within this covered region, we identified 968 nucleotide differences, 249 of which were found in coding regions. Of these differences, 238 resulted in amino acid substitutions, 98 of which were conservative and 140 of which were non-conservative as classified by the BLOSUM62 alignment score matrix. Likewise, Mesmerelda shares 95.1% GCS with its closest relative, Orfeu (Russell and Hatfull, 2017). These phages share 99.16% nucleotide identity over 100% coverage. Of the 557 nucleotide differences, 136 differences occur in coding regions. These differences result in 115 amino acid substitutions, 53 of which are conservative and 62 of which are not conservative.
Nucleotide sequence accession numbers
ElvisPhasley and Mesmerelda are available at GenBank with Accession No. PV876949 and PV876954, and Sequence Read Archive (SRA) No. SRX29990110 and SRX29990101.
Table 1: Genome and sequencing information for Mesmerelda and ElvisPhasley
Phage | ElvisPhasley | Mesmerelda |
Isolation GPS coordinates | 37° 16' 13.4034\" N 76° 43' 3.216\" W | 37° 16' 11.8992\" N 76° 42' 52.5996\" W |
Morphology | Siphovirus | Siphovirus |
Average capsid diameter (± SD) | 60 ± 4 nm (n = 5) | 50 ± 4 nm (n = 5) |
Average tail length (± SD) | 310 ± 10 nm (n = 5) | 220 ± 10 nm (n = 5) |
Sequencing reads | 4,697,092 | 4,305,175 |
Sequencing coverage, fold | 6,513 | 5,969 |
Genome length (bp) | 69,502 | 68,890 |
Character of genome ends | Circularly permuted | Circularly permuted |
Number of protein-coding genes | 102 | 102 |
GC content (%) | 66.4 | 66.4 |
Accession number | ||
SRA |
Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Bonacorsi A, Ferretti C, Di Luca M, Rindi L. 2024. Mycobacteriophages and Their Applications. Antibiotics. 13: 926.","pubmedId":"","doi":"10.3390/antibiotics13100926"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. Cresawn2011.","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.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, dynamics, and applications. Annu. Rev. Virol. 7: 37-61.","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLoS Pathog. 18: e1010602.","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Hosseiniporgham S, Sechi LA. 2022. A Review on Mycobacteriophages: From Classification to Applications. Pathogens. 11: 777.","pubmedId":"","doi":"10.3390/pathogens11070777"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. Jiang2014.","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Krumsiek J, Arnold R, Rattei T. 2007. Gepard: a rapid and sensitive tool for creating dotplots on genome scale.
","pubmedId":"","doi":"10.1093/bioinformatics/btm039"},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide. Nucleic Acids Res. 32: 11-16.","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17: 10.","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Pope WH, Jacobs Sera D. 2018. Annotation of bacteriophage genome sequences using DNA Master: An overview. Methods Mol. Biol. 1681: 217-229.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Rinehart CA, Gaffney B, Wood JD, Smith S. 2016. PECAAN: Phage Evidence Collection And Annotation Network.","pubmedId":"","doi":""},{"reference":"Russell DA. 2018. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Russell2018.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Sambrook J, Russell DW. 2006. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006: db.prot4455.","pubmedId":"","doi":"10.1101/pdb.prot4455"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long. PLoS Comput. Biol. 13: e1005595.","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, et al., Alva V. 2018. undefined. Computation Resources for Molecular Biology. 430: 2237.","pubmedId":"","doi":"https://doi.org/10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs Sera D, Freise AC, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Zorawik2024.","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"suggestedReviewer":{"name":"N/A
","WBId":""},"title":"Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda
","reviews":[]},{"id":"822a9c91-d20e-4238-8fe6-b78739b690af","decisionLetter":"\n\n Dear Authors,\n
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\n \"Figgins V, Hussey G, Haimowitz S, Pande V, Royster M, Flanagan E, et al., Saha M. 2026. Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda. microPublication Biology. 10.17912/micropub.biology.001964.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Mycobacteriophages ElvisPhasley and Mesmerelda were isolated from soil and infect Mycobacterium smegmatis. Each phage has siphovirus morphology and encodes 102 genes. Based on broader genomic similarity to actinobacteriophages, both are assigned to the B1 subcluster.
","acknowledgements":"We thank the University of Pittsburgh for genome sequencing, Old Dominion University Applied Research Center for the TEM images, the Hatfull lab, and the entire SEA-PHAGES program for their considerable ongoing support. We also thank the many institutions with SEA-PHAGES programs for their discovery and annotation of phages which made the Gepard plot in Figure 1f possible.
","authors":[{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"vafiggins@wm.edu","firstName":"Victoria","lastName":"Figgins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"gehussey@wm.edu","firstName":"Grace","lastName":"Hussey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"shaimowitz@wm.edu","firstName":"Sarah","lastName":"Haimowitz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"vpande@wm.edu","firstName":"Vera","lastName":"Pande","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"moroyster@wm.edu","firstName":"Marcus","lastName":"Royster","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egflanagan@wm.edu","firstName":"Emmery","lastName":"Flanagan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"mefountain@wm.edu","firstName":"Me'Shar'li'a","lastName":"Fountain","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egeorge@wm.edu","firstName":"Erin","lastName":"George","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zmherring@wm.edu","firstName":"Zion","lastName":"Herring","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"smjensen@wm.edu","firstName":"Scarlett","lastName":"Jensen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"jnnguyen@wm.edu","firstName":"Jenny","lastName":"Nguyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"klonufer@wm.edu","firstName":"Konur","lastName":"Onufer","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"plsmith02@wm.edu","firstName":"Phoenix","lastName":"Smith","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"bfstrong@wm.edu","firstName":"Brennan","lastName":"Strong","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zwang64@wm.edu","firstName":"Bruce","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"ayang02@wm.edu","firstName":"Abigail","lastName":"Yang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","validation","writing_reviewEditing","visualization"],"email":"hlqian@wm.edu","firstName":"Heather","lastName":"Qian","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"mssaha@wm.edu","firstName":"Margaret","lastName":"Saha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-0096-2667"}],"comments":"Dear Editor, Thank you for your comments. We agree with every one of them and have made all the requested edits/changes as described below.
1. Figure: The plaque image scalebars report 12.5 nm instead of mm.
>>>Response: We apologize for this oversight, and we have now updated the scale bar in Fig. 1a and 1b to read “12.5 mm” instead of “12.5 nm”.
2. Figure legend: Sentence 1: Change to “Plaque morphologies of ElvisPhasley (a) and MesMerelda (b). ElvisPhasley forms clear plaques with translucent halos and Mesmerelda forms clear plaques.”
>>>Response: We agree that this rephrasing of the first sentence in the figure legend improves clarity. We have now revised the opening sentence of the figure legend based on the suggested changes to read: “Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques.”
3a. Figure legend: Dot plot. “Each line of the dot plot represents a phage” is misleading given that there are many lines of differing lengths. One could say that lines form between phage genome sequences with high nucleotide identity. I think it could also be more useful to label the X and Y access with where the B1 vs B2 genomes begin and end. If you know how to read a dot plot this is obvious but some of your readers may be new to dot plots. Given the high nucleotide identity shared across all of the B genomes, I am also not sure it is helpful to label the location of Mesmerelda and ElvisPhasely in the matrix. The labels as are of these two genomes doesn’t actually make sense given that the genomes run along the X and Y access and the lines are simple nucleotide identities between the sequences on the axes.
>>>Response: We agree that our description of the Gepard dot plot was misleading and have now updated the figure legend to read: “Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences.” Additionally, we have removed the labels for ElvisPhasley and Mesmerelda on the Gepard plot because we agree that these labels are unnecessary. Finally, labels have been added to the X and Y axes of the Gepard plot to indicate which dots refer to B1 phages and which refer to B2 phages.
3b. Figure legend: Dot plot. Typo. Change “gnomes” to genomes in sentence reading, “Both aces represent all 50 phage gnomes…”
>>>Response: We apologize for missing this typo, and we have now updated the manuscript to read: “Both axes represent all 50 phage genomes…”.
4. Figure legend: genome maps. Change “Alignment of ElvisPhasely and Mesmerelda using Phamerator” to “Alignment of ElvisPhasely and Mesmerelda genomes using Phamerator.”
>>>Response: We agree that adding the word “genomes” to “Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator” in the figure legend improves clarity, and we have updated the figure legend to reflect this suggestion.
5. Paragraph 2: typo “Rsultant cultures” is misspelled.
>>>Response: Once again, we apologize for this typo and have updated Paragraph 2 of the manuscript to read: “Resultant cultures were filtered…”.
6. Paragraph 2: 0.22-uM filter is used as an adjective and therefore there should be a hyphen between number and unit.
>>>Response: We appreciate this correction, and we have now updated the text of Paragraph 2 to read: “…filtered using a 0.22-µm filter…” with the suggested hyphen between the unit and number.
7. Paragraph 2: what strain of M. smegmatis used? MC2155?
>>>Response: We have now updated both sentences referencing M. smegmatis to specify that the M. smegmatis mc2 155 strain was used instead of just the first sentence.
8. Paragraph 2: “webbed plate” is slang. Instead: “flooding plates with nearly confluent bacterial lysis”
>>>Response: We apologize for our use of slang in the manuscript, and we have updated the text in Paragraph 2 to read: “…a high titer lysate was prepared by flooding plates that exhibited nearly confluent bacterial lysis with phage buffer” per the suggested rewording.
9. Table 1: Tail length: why is a ~ used to report average tail length? That feels unneeded when reporting standard error…. Also, is the standard error being reported or standard deviation? Perhaps label rows with particle dimensions as “Average tail length” and “Average capsid diameter.”
>>>Response: We agree that including the “~” symbol in table to report average tail length is unnecessary and have therefore removed it. Additionally, we have accepted the suggestion to label the rows on Table 1 with particle dimensions as “Average tail length” and “Average capsid diameter”. Finally, we would like to confirm that we report the standard deviation of average tail length and capsid diameter, not standard error. To address this in our table, we have added (± SD) to “Average capsid diameter (± SD)” and “Average tail length (± SD)” for additional clarity.
10. Paragraph 3: “Open reading frames (ORFs) were predicted using Glimmer…etc” Do you mean protein coding genes? There are lots of ORFs in the genomes but presumably we care about the ones that potentially code for proteins? Also Glimmer and GeneMark provide an auto-annotation. Did you not refine the annotation by verifying coding potential and then selecting translational starts that meet certain criteria? You mention examining start site similarity but it is not clear how that was used to manually determine starts and if other criteria (including all coding potential) were used to predict starts. Usually starts are chosen based on their inclusion of all predicted coding potential and conservation among homologs.
>>>Response: We agree that revising the text of Paragraph 4 to read “Protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5)…” is more accurate than “Open reading frames (ORFs) were predicted…” and have updated the manuscript to reflect this change. Additionally, to provide greater clarity on how we determined the start site of each gene, we have updated the text of Paragraph 4 to read, “Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007. These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990).”
11. Paragraph 6: Sentence describing dot plot data refers to wrong panel in figure (reads 1f and should read 1e).
>>>Response: We apologize for overlooking this incorrect reference to Fig 1f instead of Fig 1e. We have relabeled the sub-figures so that the Phamerator map is now Fig 1e and the Gepard plot is Fig 1f to reflect the order in which each sub-figure is referenced in the manuscript. As such, the reference to the Gepard plot in the manuscript still reads “(Fig. 1f)”, but this is now consistent with the labeling of Figure 1 and its figure legend.
We thank the reviewer and editor for their helpful comments.
On behalf of the authors,
Margaret Saha
Professor of Applied Science
This manuscript is being submitted as part of the HHMI-microPublication workflow #HHMI_B4XYHB_2025_28.
","dataTable":null,"disclaimer":true,"funding":"","image":{"name":"260308_2018_ManuscriptFigure.jpg","url":"https://portal.micropublication.org/uploads/8326d1e514704e97e897c4c193440fb1.jpg"},"imageCaption":"Fig. 1 Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques. Scale bars for plaque images are 12.5 mm. Negative stain (uranyl acetate, 1%) transmission electron microscopy (TEM) of ElvisPhasley (c) and Mesmerelda (d) revealing siphoviral morphology. Scale bars for micrographs are 50 nm. (e) Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator. The genome is represented by the ruler, in kilo base pairs, with boxes above and below the ruler representing forward and reverse transcribed genes, respectively, and gene numbers presented within the box. (f) Gepard plot comparing the genomic sequences of 40 B1 cluster phages, including ElvisPhasley and Mesmerelda, and 10 B2 cluster phages. Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences. The Gepard plot exemplifies the genomic similarities of ElvisPhasley and Mesmerelda to other B1 cluster phages and the genomic dissimilarity of ElvisPhasley and Mesmerelda to B2 cluster phages.
","imageTitle":"Plaque and TEM Images and Genomic Organization of ElvisPhasley and Mesmerelda
","laboratory":{"name":"","WBId":""},"methods":"","reagents":"","patternDescription":"Advancing the characterization of mycobacteriophages, bacteriophages that infect Mycobacterium hosts, is of interest due to increasing antibiotic resistance in pathogenic Mycobacterium species (Hatfull, 2020; Hatfull, 2022; Bonacorsi et al., 2024). Mycobacteriophage engineering is a promising approach in the detection and treatment of mycobacterial infections (Bonacorsi et al., 2024; Hosseiniporgham et al., 2022). Therefore, the continued characterization of mycobacteriophages broadens the capabilities of bioengineers in combating this global issue. Here, we report the genome sequences of two novel mycobacteriophages, ElvisPhasley and Mesmerelda.
Phages were isolated from two different soil samples: ElvisPhasley from wet, silty soil on the William & Mary campus in Williamsburg, VA; Mesmerelda from a bag of commercially available garden soil (Table 1). A standard enrichment procedure was followed for both samples: 5 g of each sample was suspended in 45 mL of 7H9 media, inoculated with Mycobacterium smegmatis mc2 155, and incubated in a 37°C shaker at 250 rpm for 48 hours (Zorawik et al., 2024). Resultant cultures were filtered using a 0.22-µm filter, and filtrates were plated in 7H9 top agar with M. smegmatis mc2 155. After 24-48 hours, both ElvisPhasley and Mesmerelda produced clear plaques (Fig. 1a-b). Both phages were purified with three rounds of plating, and a high titer lysate was prepared by flooding plates exhibiting nearly confluent bacterial lysis with phage buffer (10 mM Tris, pH 7.5; 10 mM MgSO4; 68 mM NaCl; 1 mM CaCl2). Negative stain (1% uranyl acetate) transmission electron microscopy of each lysate revealed siphovirus morphology for both phages (Fig. 1c-d).
Phage DNA was extracted from each high titer lysate using a phenol-chloroform-isoamyl alcohol procedure and ethanol precipitation (Sambrook and Russell, 2006). DNA was prepared for sequencing using the NEB Ultra II Library Kit and sequenced using an Illumina NextSeq 1000 sequencer (single-end, 100 base read). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Newbler (v.2.9) was then used to assemble the genome and Consed (v.29) to check for completeness (Russell, 2018; Gordon et al., 1998). Sequencing data and genome characteristics are presented in Table 1.
Genome annotation was performed using DNA Master (v.5.23.6) and PECAAN (v.20221109) (Rinehart et al., 2016; Pope and Jacobs-Sera, 2018). Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007). These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990). No tRNAs were predicted using Aragorn (v.1.2.41) and tRNAscan (Laslett and Canback, 2004; Lowe and Eddy, 1997).
Predictions from HHPred (using the PDB_mmCIF70, NCBI_CD, SCOPe70, and pFAM-A databases), BLASTp, and Phamerator for highly similar genes were used to assign putative gene functions (Cresawn et al., 2011; Zimmermann et al., 2018). Both phages are assigned to cluster B, subcluster B1 using the gene content similarity (GCS) tool at the Actinobacteriophage database, PhagesDB, and clustering parameters of at least 35% GCS to actinobacteriophages (Russell and Hatfull, 2017). Default settings were used for all software.
The genomes of ElvisPhasley and Mesmerelda both encode 102 protein-coding genes, with 33 and 32 genes, respectively, assigned functions involved in virion propagation, structure, and lysis (Fig. 1e). As neither ElvisPhasley nor Mesmerelda encode a putative integrase or other proteins implicated in lysogeny, these phages are predicted to be virulent. Both ElvisPhasley and Mesmerelda display strong genomic similarity to other B1 subcluster phages and genomic dissimilarity to B2 subcluster phages as displayed on a Gepard dot plot (Fig. 1f; Krumsiek et al., 2007). ElvisPhasley shares 89.22% GCS with its closest relative, OSMaximus (Russell and Hatfull, 2017). At the nucleotide level, ElvisPhasley shares 99.00% identity with OSMaximus over 97% coverage (BLAST). Within this covered region, we identified 968 nucleotide differences, 249 of which were found in coding regions. Of these differences, 238 resulted in amino acid substitutions, 98 of which were conservative and 140 of which were non-conservative as classified by the BLOSUM62 alignment score matrix. Likewise, Mesmerelda shares 95.1% GCS with its closest relative, Orfeu (Russell and Hatfull, 2017). These phages share 99.16% nucleotide identity over 100% coverage. Of the 557 nucleotide differences, 136 differences occur in coding regions. These differences result in 115 amino acid substitutions, 53 of which are conservative and 62 of which are not conservative.
Nucleotide sequence accession numbers
ElvisPhasley and Mesmerelda are available at GenBank with Accession No. PV876949 and PV876954, and Sequence Read Archive (SRA) No. SRX29990110 and SRX29990101.
Table 1: Genome and sequencing information for Mesmerelda and ElvisPhasley
Phage | ElvisPhasley | Mesmerelda |
Isolation GPS coordinates | 37° 16' 13.4034\" N 76° 43' 3.216\" W | 37° 16' 11.8992\" N 76° 42' 52.5996\" W |
Morphology | Siphovirus | Siphovirus |
Average capsid diameter (± SD) | 60 ± 4 nm (n = 5) | 50 ± 4 nm (n = 5) |
Average tail length (± SD) | 310 ± 10 nm (n = 5) | 220 ± 10 nm (n = 5) |
Sequencing reads | 4,697,092 | 4,305,175 |
Sequencing coverage, fold | 6,513 | 5,969 |
Genome length (bp) | 69,502 | 68,890 |
Character of genome ends | Circularly permuted | Circularly permuted |
Number of protein-coding genes | 102 | 102 |
GC content (%) | 66.4 | 66.4 |
Accession number | ||
SRA |
Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Bonacorsi A, Ferretti C, Di Luca M, Rindi L. 2024. Mycobacteriophages and Their Applications. Antibiotics. 13: 926.","pubmedId":"","doi":"10.3390/antibiotics13100926"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. Cresawn2011.","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.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2020. Actinobacteriophages: Genomics, dynamics, and applications. Annu. Rev. Virol. 7: 37-61.","pubmedId":"","doi":"10.1146/annurev-virology-122019-070009"},{"reference":"Hatfull GF. 2022. Mycobacteriophages: From Petri dish to patient. PLoS Pathog. 18: e1010602.","pubmedId":"","doi":"10.1371/journal.ppat.1010602"},{"reference":"Hosseiniporgham S, Sechi LA. 2022. A Review on Mycobacteriophages: From Classification to Applications. Pathogens. 11: 777.","pubmedId":"","doi":"10.3390/pathogens11070777"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. Jiang2014.","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Krumsiek J, Arnold R, Rattei T. 2007. Gepard: a rapid and sensitive tool for creating dotplots on genome scale.
","pubmedId":"","doi":"10.1093/bioinformatics/btm039"},{"reference":"Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide. Nucleic Acids Res. 32: 11-16.","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Lowe TM, Eddy SR. 1997. tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence.
","pubmedId":"","doi":"10.1093/nar/25.5.955"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17: 10.","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Pope WH, Jacobs Sera D. 2018. Annotation of bacteriophage genome sequences using DNA Master: An overview. Methods Mol. Biol. 1681: 217-229.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Rinehart CA, Gaffney B, Wood JD, Smith S. 2016. PECAAN: Phage Evidence Collection And Annotation Network.","pubmedId":"","doi":""},{"reference":"Russell DA. 2018. Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Russell2018.","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_9"},{"reference":"Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage database.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Sambrook J, Russell DW. 2006. Purification of nucleic acids by extraction with phenol:chloroform. CSH Protoc. 2006: db.prot4455.","pubmedId":"","doi":"10.1101/pdb.prot4455"},{"reference":"Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: Resolving bacterial genome assemblies from short and long. PLoS Comput. Biol. 13: e1005595.","pubmedId":"","doi":"10.1371/journal.pcbi.1005595"},{"reference":"Zimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, et al., Alva V. 2018. undefined. Computation Resources for Molecular Biology. 430: 2237.","pubmedId":"","doi":"https://doi.org/10.1016/j.jmb.2017.12.007"},{"reference":"Zorawik M, Jacobs Sera D, Freise AC, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Zorawik2024.","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"suggestedReviewer":{"name":"N/A
","WBId":""},"title":"Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda
","reviews":[]},{"id":"0375bd65-9824-4376-9fd6-57d1de0e68f6","decisionLetter":"\n\n Dear Authors,\n
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\n \"Figgins V, Hussey G, Haimowitz S, Pande V, Royster M, Flanagan E, et al., Saha M. 2026. Sequence Analysis of Two B1 Mycobacteriophages, ElvisPhasley and Mesmerelda. microPublication Biology. 10.17912/micropub.biology.001964.\"\n
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\n ","decision":"publish","submitted":true,"abstract":"Mycobacteriophages ElvisPhasley and Mesmerelda were isolated from soil and infect Mycobacterium smegmatis. Each phage has siphovirus morphology and encodes 102 genes. Based on broader genomic similarity to actinobacteriophages, both are assigned to the B1 subcluster.
","acknowledgements":"We thank the University of Pittsburgh for genome sequencing, Old Dominion University Applied Research Center for the TEM images, the Hatfull lab, and the entire SEA-PHAGES program for their considerable ongoing support. We also thank the many institutions with SEA-PHAGES programs for their discovery and annotation of phages which made the Gepard plot in Figure 1f possible.
","authors":[{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"vafiggins@wm.edu","firstName":"Victoria","lastName":"Figgins","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"gehussey@wm.edu","firstName":"Grace","lastName":"Hussey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["dataCuration","formalAnalysis","investigation","validation","writing_originalDraft","writing_reviewEditing"],"email":"shaimowitz@wm.edu","firstName":"Sarah","lastName":"Haimowitz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"vpande@wm.edu","firstName":"Vera","lastName":"Pande","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"moroyster@wm.edu","firstName":"Marcus","lastName":"Royster","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egflanagan@wm.edu","firstName":"Emmery","lastName":"Flanagan","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"mefountain@wm.edu","firstName":"Me'Shar'li'a","lastName":"Fountain","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"egeorge@wm.edu","firstName":"Erin","lastName":"George","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zmherring@wm.edu","firstName":"Zion","lastName":"Herring","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"smjensen@wm.edu","firstName":"Scarlett","lastName":"Jensen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"jnnguyen@wm.edu","firstName":"Jenny","lastName":"Nguyen","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"klonufer@wm.edu","firstName":"Konur","lastName":"Onufer","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"sgprozik@wm.edu","firstName":"Savannah","lastName":"Prozik","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"plsmith02@wm.edu","firstName":"Phoenix","lastName":"Smith","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"bfstrong@wm.edu","firstName":"Brennan","lastName":"Strong","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"zwang64@wm.edu","firstName":"Bruce","lastName":"Wang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"ayang02@wm.edu","firstName":"Abigail","lastName":"Yang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["formalAnalysis","validation","writing_reviewEditing","visualization"],"email":"hlqian@wm.edu","firstName":"Heather","lastName":"Qian","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["William & Mary, Williamsburg, VA, US"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","project","resources","supervision","validation","visualization","writing_originalDraft","writing_reviewEditing","fundingAcquisition"],"email":"mssaha@wm.edu","firstName":"Margaret","lastName":"Saha","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-0096-2667"}],"comments":"Dear Editor, Thank you for your comments. We agree with every one of them and have made all the requested edits/changes as described below.
1. Figure: The plaque image scalebars report 12.5 nm instead of mm.
>>>Response: We apologize for this oversight, and we have now updated the scale bar in Fig. 1a and 1b to read “12.5 mm” instead of “12.5 nm”.
2. Figure legend: Sentence 1: Change to “Plaque morphologies of ElvisPhasley (a) and MesMerelda (b). ElvisPhasley forms clear plaques with translucent halos and Mesmerelda forms clear plaques.”
>>>Response: We agree that this rephrasing of the first sentence in the figure legend improves clarity. We have now revised the opening sentence of the figure legend based on the suggested changes to read: “Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques.”
3a. Figure legend: Dot plot. “Each line of the dot plot represents a phage” is misleading given that there are many lines of differing lengths. One could say that lines form between phage genome sequences with high nucleotide identity. I think it could also be more useful to label the X and Y access with where the B1 vs B2 genomes begin and end. If you know how to read a dot plot this is obvious but some of your readers may be new to dot plots. Given the high nucleotide identity shared across all of the B genomes, I am also not sure it is helpful to label the location of Mesmerelda and ElvisPhasely in the matrix. The labels as are of these two genomes doesn’t actually make sense given that the genomes run along the X and Y access and the lines are simple nucleotide identities between the sequences on the axes.
>>>Response: We agree that our description of the Gepard dot plot was misleading and have now updated the figure legend to read: “Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences.” Additionally, we have removed the labels for ElvisPhasley and Mesmerelda on the Gepard plot because we agree that these labels are unnecessary. Finally, labels have been added to the X and Y axes of the Gepard plot to indicate which dots refer to B1 phages and which refer to B2 phages.
3b. Figure legend: Dot plot. Typo. Change “gnomes” to genomes in sentence reading, “Both aces represent all 50 phage gnomes…”
>>>Response: We apologize for missing this typo, and we have now updated the manuscript to read: “Both axes represent all 50 phage genomes…”.
4. Figure legend: genome maps. Change “Alignment of ElvisPhasely and Mesmerelda using Phamerator” to “Alignment of ElvisPhasely and Mesmerelda genomes using Phamerator.”
>>>Response: We agree that adding the word “genomes” to “Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator” in the figure legend improves clarity, and we have updated the figure legend to reflect this suggestion.
5. Paragraph 2: typo “Rsultant cultures” is misspelled.
>>>Response: Once again, we apologize for this typo and have updated Paragraph 2 of the manuscript to read: “Resultant cultures were filtered…”.
6. Paragraph 2: 0.22-uM filter is used as an adjective and therefore there should be a hyphen between number and unit.
>>>Response: We appreciate this correction, and we have now updated the text of Paragraph 2 to read: “…filtered using a 0.22-µm filter…” with the suggested hyphen between the unit and number.
7. Paragraph 2: what strain of M. smegmatis used? MC2155?
>>>Response: We have now updated both sentences referencing M. smegmatis to specify that the M. smegmatis mc2 155 strain was used instead of just the first sentence.
8. Paragraph 2: “webbed plate” is slang. Instead: “flooding plates with nearly confluent bacterial lysis”
>>>Response: We apologize for our use of slang in the manuscript, and we have updated the text in Paragraph 2 to read: “…a high titer lysate was prepared by flooding plates that exhibited nearly confluent bacterial lysis with phage buffer” per the suggested rewording.
9. Table 1: Tail length: why is a ~ used to report average tail length? That feels unneeded when reporting standard error…. Also, is the standard error being reported or standard deviation? Perhaps label rows with particle dimensions as “Average tail length” and “Average capsid diameter.”
>>>Response: We agree that including the “~” symbol in table to report average tail length is unnecessary and have therefore removed it. Additionally, we have accepted the suggestion to label the rows on Table 1 with particle dimensions as “Average tail length” and “Average capsid diameter”. Finally, we would like to confirm that we report the standard deviation of average tail length and capsid diameter, not standard error. To address this in our table, we have added (± SD) to “Average capsid diameter (± SD)” and “Average tail length (± SD)” for additional clarity.
10. Paragraph 3: “Open reading frames (ORFs) were predicted using Glimmer…etc” Do you mean protein coding genes? There are lots of ORFs in the genomes but presumably we care about the ones that potentially code for proteins? Also Glimmer and GeneMark provide an auto-annotation. Did you not refine the annotation by verifying coding potential and then selecting translational starts that meet certain criteria? You mention examining start site similarity but it is not clear how that was used to manually determine starts and if other criteria (including all coding potential) were used to predict starts. Usually starts are chosen based on their inclusion of all predicted coding potential and conservation among homologs.
>>>Response: We agree that revising the text of Paragraph 4 to read “Protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5)…” is more accurate than “Open reading frames (ORFs) were predicted…” and have updated the manuscript to reflect this change. Additionally, to provide greater clarity on how we determined the start site of each gene, we have updated the text of Paragraph 4 to read, “Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007. These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990).”
11. Paragraph 6: Sentence describing dot plot data refers to wrong panel in figure (reads 1f and should read 1e).
>>>Response: We apologize for overlooking this incorrect reference to Fig 1f instead of Fig 1e. We have relabeled the sub-figures so that the Phamerator map is now Fig 1e and the Gepard plot is Fig 1f to reflect the order in which each sub-figure is referenced in the manuscript. As such, the reference to the Gepard plot in the manuscript still reads “(Fig. 1f)”, but this is now consistent with the labeling of Figure 1 and its figure legend.
We thank the reviewer and editor for their helpful comments.
On behalf of the authors,
Margaret Saha
Professor of Applied Science
This manuscript is being submitted as part of the HHMI-microPublication workflow #HHMI_B4XYHB_2025_28.
","dataTable":{"name":null,"url":null},"disclaimer":true,"funding":"","image":{"name":"260308_2018_ManuscriptFigure.jpg","url":"https://portal.micropublication.org/uploads/8326d1e514704e97e897c4c193440fb1.jpg"},"imageCaption":"Fig. 1 Plaque morphologies of ElvisPhasley (a) and Mesmerelda (b). ElvisPhasley forms clear plaques with translucent halos, and Mesmerelda forms clear plaques. Scale bars for plaque images are 12.5 mm. Negative stain (uranyl acetate, 1%) transmission electron microscopy (TEM) of ElvisPhasley (c) and Mesmerelda (d) revealing siphoviral morphology. Scale bars for micrographs are 50 nm. (e) Alignment of ElvisPhasley and Mesmerelda genomes using Phamerator. The genome is represented by the ruler, in kilo base pairs, with boxes above and below the ruler representing forward and reverse transcribed genes, respectively, and gene numbers presented within the box. (f) Gepard plot comparing the genomic sequences of 40 B1 cluster phages, including ElvisPhasley and Mesmerelda, and 10 B2 cluster phages. Both axes represent all 50 phage genomes, and each diagonal line on the dot plot represents high nucleotide identity between phage genome sequences. The Gepard plot exemplifies the genomic similarities of ElvisPhasley and Mesmerelda to other B1 cluster phages and the genomic dissimilarity of ElvisPhasley and Mesmerelda to B2 cluster phages.
","imageTitle":"Plaque and TEM Images and Genomic Organization of ElvisPhasley and Mesmerelda
","laboratory":{"name":"","WBId":""},"methods":"","reagents":"","patternDescription":"Advancing the characterization of mycobacteriophages, bacteriophages that infect Mycobacterium hosts, is of interest due to increasing antibiotic resistance in pathogenic Mycobacterium species (Hatfull, 2020; Hatfull, 2022; Bonacorsi et al., 2024). Mycobacteriophage engineering is a promising approach in the detection and treatment of mycobacterial infections (Bonacorsi et al., 2024; Hosseiniporgham et al., 2022). Therefore, the continued characterization of mycobacteriophages broadens the capabilities of bioengineers in combating this global issue. Here, we report the genome sequences of two novel mycobacteriophages, ElvisPhasley and Mesmerelda.
Phages were isolated from two different soil samples: ElvisPhasley from wet, silty soil on the William & Mary campus in Williamsburg, VA; Mesmerelda from a bag of commercially available garden soil (Table 1). A standard enrichment procedure was followed for both samples: 5 g of each sample was suspended in 45 mL of 7H9 media, inoculated with Mycobacterium smegmatis mc2 155, and incubated in a 37°C shaker at 250 rpm for 48 hours (Zorawik et al., 2024). Resultant cultures were filtered using a 0.22-µm filter, and filtrates were plated in 7H9 top agar with M. smegmatis mc2 155. After 24-48 hours, both ElvisPhasley and Mesmerelda produced clear plaques (Fig. 1a-b). Both phages were purified with three rounds of plating, and a high titer lysate was prepared by flooding plates exhibiting nearly confluent bacterial lysis with phage buffer (10 mM Tris, pH 7.5; 10 mM MgSO4; 68 mM NaCl; 1 mM CaCl2). Negative stain (1% uranyl acetate) transmission electron microscopy of each lysate revealed siphovirus morphology for both phages (Fig. 1c-d).
Phage DNA was extracted from each high titer lysate using a phenol-chloroform-isoamyl alcohol procedure and ethanol precipitation (Sambrook and Russell, 2006). DNA was prepared for sequencing using the NEB Ultra II Library Kit and sequenced using an Illumina NextSeq 1000 sequencer (single-end, 100 base read). Raw reads were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) (Martin, 2011; Jiang et al., 2014; Wick et al., 2017). Newbler (v.2.9) was then used to assemble the genome and Consed (v.29) to check for completeness (Russell, 2018; Gordon et al., 1998). Sequencing data and genome characteristics are presented in Table 1.
Genome annotation was performed using DNA Master (v.5.23.6) and PECAAN (v.20221109) (Rinehart et al., 2016; Pope and Jacobs-Sera, 2018). Start sites of protein coding genes were predicted using Glimmer (v.3.02) and GeneMark (v.2.5) (Besemer and Borodovsky, 2005; Delcher et al., 2007). These predictions were manually refined using GeneMark coding potential predictions (Besemer and Borodovsky, 2005), start site similarity comparisons between pham members obtained from Starterator (http://phages.wustl.edu/starterator/), and start site alignment scores with homologous genes found using BLASTp against the NCBI non-redundant protein and Actinobacteriophage databases (Altschul et al., 1990). No tRNAs were predicted using Aragorn (v.1.2.41) and tRNAscan (Laslett and Canback, 2004; Lowe and Eddy, 1997).
Predictions from HHPred (using the PDB_mmCIF70, NCBI_CD, SCOPe70, and pFAM-A databases), BLASTp, and Phamerator for highly similar genes were used to assign putative gene functions (Cresawn et al., 2011; Zimmermann et al., 2018). Both phages are assigned to cluster B, subcluster B1 using the gene content similarity (GCS) tool at the Actinobacteriophage database, PhagesDB, and clustering parameters of at least 35% GCS to actinobacteriophages (Russell and Hatfull, 2017). Default settings were used for all software.
The genomes of ElvisPhasley and Mesmerelda both encode 102 protein-coding genes, with 33 and 32 genes, respectively, assigned functions involved in virion propagation, structure, and lysis (Fig. 1e). As neither ElvisPhasley nor Mesmerelda encode a putative integrase or other proteins implicated in lysogeny, these phages are predicted to be virulent. Both ElvisPhasley and Mesmerelda display strong genomic similarity to other B1 subcluster phages and genomic dissimilarity to B2 subcluster phages as displayed on a Gepard dot plot (Fig. 1f; Krumsiek et al., 2007). ElvisPhasley shares 89.22% GCS with its closest relative, OSMaximus (Russell and Hatfull, 2017). At the nucleotide level, ElvisPhasley shares 99.00% identity with OSMaximus over 97% coverage (BLAST). Within this covered region, we identified 968 nucleotide differences, 249 of which were found in coding regions. Of these differences, 238 resulted in amino acid substitutions, 98 of which were conservative and 140 of which were non-conservative as classified by the BLOSUM62 alignment score matrix. Likewise, Mesmerelda shares 95.1% GCS with its closest relative, Orfeu (Russell and Hatfull, 2017). These phages share 99.16% nucleotide identity over 100% coverage. Of the 557 nucleotide differences, 136 differences occur in coding regions. These differences result in 115 amino acid substitutions, 53 of which are conservative and 62 of which are not conservative.
Nucleotide sequence accession numbers
ElvisPhasley and Mesmerelda are available at GenBank with Accession No. PV876949 and PV876954, and Sequence Read Archive (SRA) No. SRX29990110 and SRX29990101.
Table 1: Genome and sequencing information for Mesmerelda and ElvisPhasley
Phage | ElvisPhasley | Mesmerelda |
Isolation GPS coordinates | 37° 16' 13.4034\" N 76° 43' 3.216\" W | 37° 16' 11.8992\" N 76° 42' 52.5996\" W |
Morphology | Siphovirus | Siphovirus |
Average capsid diameter (± SD) | 60 ± 4 nm (n = 5) | 50 ± 4 nm (n = 5) |
Average tail length (± SD) | 310 ± 10 nm (n = 5) | 220 ± 10 nm (n = 5) |
Sequencing reads | 4,697,092 | 4,305,175 |
Sequencing coverage, fold | 6,513 | 5,969 |
Genome length (bp) | 69,502 | 68,890 |
Character of genome ends | Circularly permuted | Circularly permuted |
Number of protein-coding genes | 102 | 102 |
GC content (%) | 66.4 | 66.4 |
Accession number | ||
SRA |
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