Actinobacteriophage BaronJohn was isolated from soil collected near the Lakeside dormitory building on the campus of the College of Coastal Georgia using Microbacterium foliorum NRRL B-24224 as a host. BaronJohn has a genome length and gene content similar to that of phages in cluster EA5. Specifically, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a tRNA-Ala gene, two glycosyltransferases in tandem and a lysin A downstream of the minor tail proteins.
","acknowledgements":"We thank the SEA-PHAGES program and Howard Hughes Medical Institute for supporting this research. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena and Colleen Knight for their technical support. We thank the SEA MINT team for their thorough review of this manuscript.
","authors":[{"affiliations":["University of Georgia, Athens, Georgia USA"],"departments":["College of Public Health"],"credit":["investigating","writing_reviewEditing"],"email":"John.Barron@uga.edu","firstName":"John","lastName":"Barron","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920156676@student.ccga.edu","firstName":"John","lastName":"Barton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920055698@student.ccga.edu","firstName":"Brittni","lastName":"Brown","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149231@student.ccga.edu","firstName":"Madeleine","lastName":"Culbreath","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920147718@student.ccga.edu","firstName":"Phoebe Gerlyn","lastName":"De Jesus ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920137401@student.ccga.edu","firstName":"Elena","lastName":"Doppel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920158442@student.ccga.edu","firstName":"Paige","lastName":"Gailey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920143290@student.ccga.edu","firstName":"Brenda","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149421@student.ccga.edu","firstName":"Mark","lastName":"Quarterman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920164802@student.ccga.edu","firstName":"Kylie","lastName":"Thomas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920148457@student.ccga.edu","firstName":"Joanna","lastName":"Thomason","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920116221@student.ccga.edu","firstName":"Felicia","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920141727@student.ccga.edu","firstName":"Kevin","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["supervision","validation","investigation","visualization","writingOriginalDraftWriting","writing_reviewEditing"],"email":"hnance@ccga.edu","firstName":"Holly","lastName":"Nance","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"We thank the College of Coastal Georgia Department of Natural Sciences for supporting this work.
","image":{"url":"https://portal.micropublication.org/uploads/bcc90766848cb3a782f59606c5a39f59.png"},"imageCaption":"Genes are presented as boxes along a genome ruler (in kilobases), with different colors representing different gene families based on sequence similarity. Boxes above the ruler represent genes that are transcribed in the forward direction and genes below the ruler are transcribed in the reverse direction. All predicted gene functions are labeled.
","imageTitle":"Phamerator map of BaronJohn’s genome
","methods":"","reagents":"","patternDescription":"As the most abundant biological lifeforms on the planet, bacteriophages that infect a bacterial host provide critical roles within microbes and help regulate their populations (Hatfull 2015). Characterizing newly isolated bacteriophages expands not only our knowledge, but it may also reveal novel genes within their genomes that hold biological significance for our species (Bailly-Bechet et al. 2007). This knowledge can provide insight into the ever-expanding host-virus dynamic and theoretically give rise to therapeutic clinical applications, for example to isolate and evaluate bacteria with pathogenic specificity to combat potential diseases (Pope et al. 2015). As antibiotic resistance continues to rise, phage therapy has gained renewed interest as a potential alternative treatment. Due to their high specificity and ability to lyse bacterial cells, bacteriophages may offer a promising solution to multidrug-resistant infections (Zalewska-Piątek 2023).
The Microbacterium phage BaronJohn was isolated from a soil sample collected in Brunswick, GA (31°10'56\" N, 81° 21' 59\" W) using an enriched isolation protocol (Zorawik et al. 2024). The soil sample was incubated in a peptone-yeast-calcium (PYCa) liquid medium at 32°C while shaking for two hours. After incubation, the sample was centrifuged at 2,000 x g for 10 minutes and the supernatant was filtered through a 0.22 µm pore size. This supernatant was collected in a vented 50 ml conical tube, seeded with 0.5 ml of Microbacterium foliorum, and incubated at 32°C with shaking for three days. Following incubation, the supernatant was filtered through a 0.22 µm pore size membrane, and serial dilutions (10-1 to 10-8 µl) were plated with host M. foliorum on PYCa medium with soft agar overlay. After 48 hours of incubation at 32°C, clear, well-defined plaques were observed. BaronJohn was purified through four rounds of plaque assays.
Phage DNA was extracted from lysate. Briefly, bacterial DNA and RNA in the lysate was first degraded by treatment with DNase I and RNaseA. Nucleases were subsequently inactivated with EDTA, Proteinase K, and SDS, after which phage DNA was extracted using the Promega Wizard DNA Cleanup System. Libraries were prepped using the NEB II Ultra Library kit and sequenced using Illumina NextSeq 1000, producing 2,501,030 single-end 100-bp reads (5077-fold coverage). Raw reads were trimmed with Cutadapt 4.7 (Miller 2011), using the option: –nextseq-trim 30, and filtered with Skewer 0.2.2 (Jiang et al. 2014), using the options: -q 20 -Q 30 -n -l 50, prior to assembly. BaronJohn’s genomic termini were assessed using Consed v29 (Gordon and Green 2013) following Russell (2018) and raw reads were assembled using Newbler v2.9 (Miller et al. 2010). The genome of BaronJohn is 40,502 base pairs long and circularly permuted with a G+C content of 63.9%, which is similar to the 68.7% G+C content of host M. foliorum (Darley et al. 2026).
Glimmer v3.02 (Delcher et al. 2007) and GeneMark.hmm v3.36 (Besemer and Borodovsky 2005) were used in the initial auto-annotation to identify regions with coding potential. DNA Master v5.23.6 (Pope and Jacobs-Sera 2017) and Starterator v587 (http://phages.wustl.edu/starterator/) were used to review these predictions and revise as needed. BLASTp v2.2.26 (Altschul et al. 1990) with the Actinobacteriophage and NCBI non-redundant databases and PhagesDB (Russell and Hatfull 2016) were used to compare sequences and functions of similar genes. Phamerator, using Actino-draft database v643 (Cresawn et al. 2011) was used to compare gene location, length, and conserved domains among closely related phages. HHpred MPI Bioinformatics Toolkit with databases PDB_mmCIF70_20_Feb, SCOPe70_2.08, Pfam-A_v38.2, NCBI_Conserved_Domains (CD)_v3.19, and UniProt-SwissProt-viral70_3_Nov_2021 was used to analyze protein crystalline structure to predict function (Söding et al. 2005). DeepTMHMM v1.044 (Chen et al. 2003) was used to identify transmembrane proteins. Aragorn v1.2.41 was used to confirm the presence and location of tRNA genes (Laslett and Canback 2004). Default settings were used for all software.
BaronJohn was assigned to subcluster EA5 due to at least 35% shared gene content similarity to phages in the Actinobacteriophage database (https://phagesDB.org; Russell and Hatfull 2016; Pope et al. 2017). The G+C content of 63.9% falls within the range (63.5-64.4%) for subcluster EA5 phages. BaronJohnhas a total of 61 genes, 24 of which were assigned functions, and one tRNA-Ala gene (see Fig. 1 for details). This is consistent with the 15 other phages in subcluster EA5 to date, whose number of genes range from 57-63 per genome, and 10 of 15 phages which also have a tRNA gene that is either Ala or undetermined, and syntenic (https://phagesDB.org). Five of the genes in BaronJohn were found to contain transmembrane domains, although the function could not be determined. Roughly half of the genes (28) are transcribed in a single direction, with the other half (33) transcribed from the opposite strand. Like all other EA5 phages, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a MazG-like nucleotide pyrophosphohydrolase, and two glycosyltransferases (all EA5 phages annotated to date have either one or two in tandem). All 16 EA5 phages encode a lysin A (previously called endolysin) downstream of the minor tail proteins, although these genes comprise two different gene families (Phamilies). No genes canonically associated with lysogeny (e.g. integrase, immunity repressor) were identified on this genome, suggesting a virulent (lytic) life cycle.
Nucleotide Accession Number
GenBank Accession Number is PZ575424. The Sequence Read Archive (SRA) number is SRX32199476.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research 17: 1486-1495.
","pubmedId":"","doi":"10.1101/gr.6649807"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Darley K, English N, Barron J, Brooks E, Brown K, Claxton A, et al., Nance H. 2026. Genome Sequence of Actinobacteriophage LilTerminator, Isolated from Microbacterium foliorum on Jekyll Island, Georgia. microPublication Biology.
","pubmedId":"","doi":""},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.
","pubmedId":"","doi":"10.1128/JVI.01340-15"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. In M. Clokie, A. Kropinski, & R. Lavigne (Eds.), Bacteriophages (Vol. 1681, pp. 109–125). Humana Press.
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zalewska-Piątek B. 2023. Phage Therapy—Challenges, Opportunities and Future Prospects. Pharmaceuticals 16: 1638.
","pubmedId":"","doi":" 10.3390/ph16121638"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence of Actinobacteriophage BaronJohn, Isolated on Microbacterium foliorum on the College of Coastal Georgia Campus
","reviews":[{"reviewer":{"displayName":"Frederick N Baliraine"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"7563d914-1222-453a-be60-2de63b84f1b6","decision":"accept","abstract":"Actinobacteriophage BaronJohn was isolated from soil collected near the Lakeside dormitory building on the campus of the College of Coastal Georgia using Microbacterium foliorum NRRL B-24224 as a host. BaronJohn has a genome length and gene content similar to that of phages in cluster EA5. Specifically, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a tRNA-Ala gene, two glycosyltransferases in tandem and a lysin A downstream of the minor tail proteins.
","acknowledgements":"We thank the SEA-PHAGES program and Howard Hughes Medical Institute for supporting this research. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena and Colleen Knight for their technical support. We thank the SEA MINT team for their thorough review of this manuscript.
","authors":[{"affiliations":["University of Georgia, Athens, Georgia USA"],"departments":["College of Public Health"],"credit":["investigating","writing_reviewEditing"],"email":"John.Barron@uga.edu","firstName":"John","lastName":"Barron","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920156676@student.ccga.edu","firstName":"John","lastName":"Barton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920055698@student.ccga.edu","firstName":"Brittni","lastName":"Brown","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149231@student.ccga.edu","firstName":"Madeleine","lastName":"Culbreath","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920147718@student.ccga.edu","firstName":"Phoebe Gerlyn","lastName":"De Jesus ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920137401@student.ccga.edu","firstName":"Elena","lastName":"Doppel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920158442@student.ccga.edu","firstName":"Paige","lastName":"Gailey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920143290@student.ccga.edu","firstName":"Brenda","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149421@student.ccga.edu","firstName":"Mark","lastName":"Quarterman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920164802@student.ccga.edu","firstName":"Kylie","lastName":"Thomas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920148457@student.ccga.edu","firstName":"Joanna","lastName":"Thomason","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920116221@student.ccga.edu","firstName":"Felicia","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920141727@student.ccga.edu","firstName":"Kevin","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["supervision","validation","investigation","visualization","writingOriginalDraftWriting","writing_reviewEditing"],"email":"hnance@ccga.edu","firstName":"Holly","lastName":"Nance","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"We thank the College of Coastal Georgia Department of Natural Sciences for supporting this work.
","image":{"url":"https://portal.micropublication.org/uploads/bcc90766848cb3a782f59606c5a39f59.png"},"imageCaption":"Genes are presented as boxes along a genome ruler (in kilobases), with different colors representing different gene families based on sequence similarity. Boxes above the ruler represent genes that are transcribed in the forward direction and genes below the ruler are transcribed in the reverse direction. All predicted gene functions are labeled.
","imageTitle":"Phamerator map of BaronJohn’s genome
","methods":"","reagents":"","patternDescription":"As the most abundant biological lifeforms on the planet, bacteriophages provide critical roles within microbes and help regulate their populations (Hatfull 2015). Characterizing newly isolated bacteriophages expands not only our knowledge, but it may also reveal novel genes within their genomes that hold biological significance for our species (Bailly-Bechet et al. 2007). This knowledge can provide insight into the ever-expanding host-virus dynamic and theoretically give rise to therapeutic clinical applications, for example to isolate and evaluate bacteria with pathogenic specificity to combat potential diseases (Pope et al. 2015). As antibiotic resistance continues to rise, phage therapy has gained renewed interest as a potential alternative treatment. Due to their high specificity and ability to lyse bacterial cells, bacteriophages may offer a promising solution to multidrug-resistant infections (Zalewska-Piątek 2023).
The Microbacterium phage BaronJohn was isolated from a soil sample collected in Brunswick, GA (31°10'56\" N, 81° 21' 59\" W) using an enriched isolation protocol (Zorawik et al. 2024). The soil sample was incubated in a peptone-yeast-calcium (PYCa) liquid medium at 32°C while shaking for two hours at 360 RPM using a Labnet Orbit™ 300 Digital Multipurpose Shaker. After incubation, the sample was centrifuged at 2,000 x g for 10 minutes and the supernatant was filtered through a 0.22 µm pore size. This supernatant was collected in a vented 50 ml conical tube, seeded with 0.5 ml of Microbacterium foliorum, and incubated at 32°C with shaking at 360 RPM for three days. Following incubation, the supernatant was filtered through a 0.22 µm pore size membrane, and serial dilutions (10-1 to 10-8 µl) were plated with host M. foliorum on PYCa medium with soft agar overlay. After 48 hours of incubation at 32°C, clear, well-defined plaques were observed. BaronJohn was purified through four rounds of plaque assays.
Phage DNA was extracted from high-titer lysate (6 x 1010 pfu/ml). Briefly, bacterial DNA and RNA in the lysate was first degraded by treatment with DNase I and RNaseA. Nucleases were subsequently inactivated with EDTA, Proteinase K, and SDS, after which phage DNA was extracted using the Promega Wizard DNA Cleanup System. Libraries were prepped using the NEB II Ultra Library kit and sequenced using Illumina NextSeq 1000, producing 2,501,030 single-end 100-bp reads (5077-fold coverage). Raw reads were trimmed with Cutadapt 4.7 (Miller 2011), using the option: –nextseq-trim 30, and filtered with Skewer 0.2.2 (Jiang et al. 2014), using the options: -q 20 -Q 30 -n -l 50, prior to assembly. BaronJohn’s genomic termini were assessed using Consed v29 (Gordon and Green 2013) following Russell (2018) and raw reads were assembled using Newbler v2.9 (Miller et al. 2010). The genome of BaronJohn is 40,502 base pairs long and circularly permuted with a G+C content of 63.9%.
Glimmer v3.02 (Delcher et al. 2007) and GeneMark.hmm v3.36 (Besemer and Borodovsky 2005) were used in the initial auto-annotation to identify regions with coding potential. DNA Master v5.23.6 (Pope and Jacobs-Sera 2017) and Starterator v587 (http://phages.wustl.edu/starterator/) were used to review these predictions and revise as needed. BLASTp v2.2.26 (Altschul et al. 1990) with the Actinobacteriophage and NCBI non-redundant databases and PhagesDB (Russell and Hatfull 2016) were used to compare sequences and functions of similar genes. Phamerator, using Actino-draft database v643 (Cresawn et al. 2011) was used to compare gene location, length, and conserved domains among closely related phages. HHpred MPI Bioinformatics Toolkit with databases PDB_mmCIF70_20_Feb, SCOPe70_2.08, Pfam-A_v38.2, NCBI_Conserved_Domains (CD)_v3.19, and UniProt-SwissProt-viral70_3_Nov_2021 was used to analyze protein crystalline structure to predict function (Söding et al. 2005). DeepTMHMM v1.044 (Chen et al. 2003) was used to identify transmembrane proteins. Aragorn v1.2.41 was used to confirm the presence and location of tRNA genes (Laslett and Canback 2004). Default settings were used for all software.
BaronJohn was assigned to subcluster EA5 due to at least 35% shared gene content similarity to phages in the Actinobacteriophage database (https://phagesDB.org; Russell and Hatfull 2016; Pope et al. 2017). The G+C content of 63.9% falls within the range (63.5-64.4%) for subcluster EA5 phages. BaronJohnhas a total of 61 putative genes, 24 of which were assigned functions, and one tRNA-Ala gene (see Fig. 1 for details). This is consistent with the 15 other phages in subcluster EA5 to date, whose number of genes range from 57-63 per genome, and 10 of 15 phages which also have a tRNA gene that is either Ala or undetermined, and syntenic (https://phagesDB.org). Five of the genes in BaronJohn were found to contain transmembrane domains, although the function could not be determined. Roughly half of the genes (28) are transcribed in the forward direction, with the other half (33) transcribed from the opposite strand. Like all other EA5 phages, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a MazG-like nucleotide pyrophosphohydrolase, and two glycosyltransferases (all EA5 phages annotated to date have either one or two in tandem). All 16 EA5 phages encode a lysin A (previously called endolysin) downstream of the minor tail proteins, although these genes comprise two different gene families (Phamilies). No genes canonically associated with lysogeny (e.g. integrase, immunity repressor) were identified on this genome, suggesting a virulent (lytic) life cycle.
Nucleotide Accession Number
GenBank Accession Number is PZ575424. The Sequence Read Archive (SRA) number is SRX32199476.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research 17: 1486-1495.
","pubmedId":"","doi":"10.1101/gr.6649807"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.
","pubmedId":"","doi":"10.1128/JVI.01340-15"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. In M. Clokie, A. Kropinski, & R. Lavigne (Eds.), Bacteriophages (Vol. 1681, pp. 109–125). Humana Press.
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zalewska-Piątek B. 2023. Phage Therapy—Challenges, Opportunities and Future Prospects. Pharmaceuticals 16: 1638.
","pubmedId":"","doi":" 10.3390/ph16121638"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence of Actinobacteriophage BaronJohn, Isolated on Microbacterium foliorum on the College of Coastal Georgia Campus
","reviews":[],"curatorReviews":[]},{"id":"2f340092-087a-46d0-ab99-caff0cc5f03a","decision":"publish","abstract":"Actinobacteriophage BaronJohn was isolated from soil collected near the Lakeside dormitory building on the campus of the College of Coastal Georgia using Microbacterium foliorum NRRL B-24224 as a host. BaronJohn has a genome length and gene content similar to that of phages in cluster EA5. Specifically, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a tRNA-Ala gene, two glycosyltransferases in tandem and a lysin A downstream of the minor tail proteins.
","acknowledgements":"We thank the SEA-PHAGES program and Howard Hughes Medical Institute for supporting this research. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena and Colleen Knight for their technical support. We thank the SEA MINT team for their thorough review of this manuscript.
","authors":[{"affiliations":["University of Georgia, Athens, Georgia USA"],"departments":["College of Public Health"],"credit":["investigating","writing_reviewEditing"],"email":"John.Barron@uga.edu","firstName":"John","lastName":"Barron","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920156676@student.ccga.edu","firstName":"John","lastName":"Barton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920055698@student.ccga.edu","firstName":"Brittni","lastName":"Brown","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149231@student.ccga.edu","firstName":"Madeleine","lastName":"Culbreath","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920147718@student.ccga.edu","firstName":"Phoebe Gerlyn","lastName":"De Jesus ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920137401@student.ccga.edu","firstName":"Elena","lastName":"Doppel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920158442@student.ccga.edu","firstName":"Paige","lastName":"Gailey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920143290@student.ccga.edu","firstName":"Brenda","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920149421@student.ccga.edu","firstName":"Mark","lastName":"Quarterman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920164802@student.ccga.edu","firstName":"Kylie","lastName":"Thomas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920148457@student.ccga.edu","firstName":"Joanna","lastName":"Thomason","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920116221@student.ccga.edu","firstName":"Felicia","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","validating","writingOriginalDraftWriting"],"email":"920141727@student.ccga.edu","firstName":"Kevin","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["supervision","validation","investigation","visualization","writingOriginalDraftWriting","writing_reviewEditing"],"email":"hnance@ccga.edu","firstName":"Holly","lastName":"Nance","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"We thank the College of Coastal Georgia Department of Natural Sciences for supporting this work.
","image":{"url":"https://portal.micropublication.org/uploads/bcc90766848cb3a782f59606c5a39f59.png"},"imageCaption":"Genes are presented as boxes along a genome ruler (in kilobases), with different colors representing different gene families based on sequence similarity. Boxes above the ruler represent genes that are transcribed in the forward direction and genes below the ruler are transcribed in the reverse direction. All predicted gene functions are labeled.
","imageTitle":"Phamerator map of BaronJohn’s genome
","methods":"","reagents":"","patternDescription":"As the most abundant biological lifeforms on the planet, bacteriophages provide critical roles within microbes and help regulate their populations (Hatfull 2015). Characterizing newly isolated bacteriophages expands not only our knowledge, but it may also reveal novel genes within their genomes that hold biological significance for our species (Bailly-Bechet et al. 2007). This knowledge can provide insight into the ever-expanding host-virus dynamic and theoretically give rise to therapeutic clinical applications, for example to isolate and evaluate bacteria with pathogenic specificity to combat potential diseases (Pope et al. 2015). As antibiotic resistance continues to rise, phage therapy has gained renewed interest as a potential alternative treatment. Due to their high specificity and ability to lyse bacterial cells, bacteriophages may offer a promising solution to multidrug-resistant infections (Zalewska-Piątek 2023).
The Microbacterium phage BaronJohn was isolated from a soil sample collected in Brunswick, GA (31°10'56\" N, 81° 21' 59\" W) using an enriched isolation protocol (Zorawik et al. 2024). The soil sample was incubated in a peptone-yeast-calcium (PYCa) liquid medium at 32°C while shaking for two hours at 360 RPM using a Labnet Orbit™ 300 Digital Multipurpose Shaker. After incubation, the sample was centrifuged at 2,000 x g for 10 minutes and the supernatant was filtered through a 0.22 µm pore size. This supernatant was collected in a vented 50 ml conical tube, seeded with 0.5 ml of Microbacterium foliorum, and incubated at 32°C with shaking at 360 RPM for three days. Following incubation, the supernatant was filtered through a 0.22 µm pore size membrane, and serial dilutions (10-1 to 10-8 µl) were plated with host M. foliorum on PYCa medium with soft agar overlay. After 48 hours of incubation at 32°C, clear, well-defined plaques were observed. BaronJohn was purified through four rounds of plaque assays.
Phage DNA was extracted from high-titer lysate (6 x 1010 pfu/ml). Briefly, bacterial DNA and RNA in the lysate was first degraded by treatment with DNase I and RNaseA. Nucleases were subsequently inactivated with EDTA, Proteinase K, and SDS, after which phage DNA was extracted using the Promega Wizard DNA Cleanup System. Libraries were prepped using the NEB II Ultra Library kit and sequenced using Illumina NextSeq 1000, producing 2,501,030 single-end 100-bp reads (5077-fold coverage). Raw reads were trimmed with Cutadapt 4.7 (Miller 2011), using the option: –nextseq-trim 30, and filtered with Skewer 0.2.2 (Jiang et al. 2014), using the options: -q 20 -Q 30 -n -l 50, prior to assembly. BaronJohn's genomic termini were assessed using Consed v29 (Gordon and Green 2013) following Russell (2018) and raw reads were assembled using Newbler v2.9 (Miller et al. 2010). The genome of BaronJohn is 40,502 base pairs long and circularly permuted with a G+C content of 63.9%.
Glimmer v3.02 (Delcher et al. 2007) and GeneMark.hmm v3.36 (Besemer and Borodovsky 2005) were used in the initial auto-annotation to identify regions with coding potential. DNA Master v5.23.6 (Pope and Jacobs-Sera 2017) and Starterator v587 (http://phages.wustl.edu/starterator/) were used to review these predictions and revise as needed. BLASTp v2.2.26 (Altschul et al. 1990) with the Actinobacteriophage and NCBI non-redundant databases and PhagesDB (Russell and Hatfull 2016) were used to compare sequences and functions of similar genes. Phamerator, using Actino-draft database v643 (Cresawn et al. 2011) was used to compare gene location, length, and conserved domains among closely related phages. HHpred MPI Bioinformatics Toolkit with databases PDB_mmCIF70_20_Feb, SCOPe70_2.08, Pfam-A_v38.2, NCBI_Conserved_Domains (CD)_v3.19, and UniProt-SwissProt-viral70_3_Nov_2021 was used to analyze protein crystalline structure to predict function (Söding et al. 2005). DeepTMHMM v1.044 (Chen et al. 2003) was used to identify transmembrane proteins. Aragorn v1.2.41 was used to confirm the presence and location of tRNA genes (Laslett and Canback 2004). Default settings were used for all software.
BaronJohn was assigned to subcluster EA5 due to at least 35% shared gene content similarity to phages in the Actinobacteriophage database (https://phagesDB.org; Russell and Hatfull 2016; Pope et al. 2017). The G+C content of 63.9% falls within the range (63.5-64.4%) for subcluster EA5 phages. BaronJohnhas a total of 61 putative genes, 24 of which were assigned functions, and one tRNA-Ala gene (see Fig. 1 for details). This is consistent with the 15 other phages in subcluster EA5 to date, whose number of genes range from 57-63 per genome, and 10 of 15 phages which also have a tRNA gene that is either Ala or undetermined, and syntenic (https://phagesDB.org). Five of the genes in BaronJohn were found to contain transmembrane domains, although the function could not be determined. Roughly half of the genes (28) are transcribed in the forward direction, with the other half (33) transcribed from the opposite strand. Like all other EA5 phages, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a MazG-like nucleotide pyrophosphohydrolase, and two glycosyltransferases (all EA5 phages annotated to date have either one or two in tandem). All 16 EA5 phages encode a lysin A (previously called endolysin) downstream of the minor tail proteins, although these genes comprise two different gene families (Phamilies). No genes canonically associated with lysogeny (e.g. integrase, immunity repressor) were identified on this genome, suggesting a virulent (lytic) life cycle.
Nucleotide Accession Number
GenBank Accession Number is PZ575424. The Sequence Read Archive (SRA) number is SRX32199476.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research 17: 1486-1495.
","pubmedId":"","doi":"10.1101/gr.6649807"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.
","pubmedId":"","doi":"10.1128/JVI.01340-15"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. In M. Clokie, A. Kropinski, & R. Lavigne (Eds.), Bacteriophages (Vol. 1681, pp. 109–125). Humana Press.
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zalewska-Piątek B. 2023. Phage Therapy—Challenges, Opportunities and Future Prospects. Pharmaceuticals 16: 1638.
","pubmedId":"","doi":" 10.3390/ph16121638"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence of Actinobacteriophage BaronJohn, Isolated on Microbacterium foliorum on the College of Coastal Georgia Campus
","reviews":[],"curatorReviews":[]},{"id":"58bb01f4-d85c-4be1-8aee-3d9251c9cfca","decision":"publish","abstract":"Actinobacteriophage BaronJohn was isolated from soil collected near the Lakeside dormitory building on the campus of the College of Coastal Georgia using Microbacterium foliorum NRRL B-24224 as a host. BaronJohn has a genome length and gene content similar to that of phages in cluster EA5. Specifically, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a tRNA-Ala gene, two glycosyltransferases in tandem and a lysin A downstream of the minor tail proteins.
","acknowledgements":"We thank the SEA-PHAGES program and Howard Hughes Medical Institute for supporting this research. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena and Colleen Knight for their technical support. We thank the SEA MINT team for their thorough review of this manuscript.
","authors":[{"affiliations":["University of Georgia, Athens, Georgia USA"],"departments":["College of Public Health"],"credit":["investigating","writing_reviewEditing"],"email":"John.Barron@uga.edu","firstName":"John","lastName":"Barron","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920156676@student.ccga.edu","firstName":"John","lastName":"Barton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920055698@student.ccga.edu","firstName":"Brittni","lastName":"Brown","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920149231@student.ccga.edu","firstName":"Madeleine","lastName":"Culbreath","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920147718@student.ccga.edu","firstName":"Phoebe Gerlyn","lastName":"De Jesus ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920137401@student.ccga.edu","firstName":"Elena","lastName":"Doppel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920158442@student.ccga.edu","firstName":"Paige","lastName":"Gailey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920143290@student.ccga.edu","firstName":"Brenda","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920149421@student.ccga.edu","firstName":"Mark","lastName":"Quarterman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920164802@student.ccga.edu","firstName":"Kylie","lastName":"Thomas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920148457@student.ccga.edu","firstName":"Joanna","lastName":"Thomason","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920116221@student.ccga.edu","firstName":"Felicia","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920141727@student.ccga.edu","firstName":"Kevin","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing","writing_originalDraft"],"email":"hnance@ccga.edu","firstName":"Holly","lastName":"Nance","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"We thank the College of Coastal Georgia Department of Natural Sciences for supporting this work.
","image":{"url":"https://portal.micropublication.org/uploads/bcc90766848cb3a782f59606c5a39f59.png"},"imageCaption":"Genes are presented as boxes along a genome ruler (in kilobases), with different colors representing different gene families based on sequence similarity. Boxes above the ruler represent genes that are transcribed in the forward direction and genes below the ruler are transcribed in the reverse direction. All predicted gene functions are labeled.
","imageTitle":"Phamerator map of BaronJohn’s genome
","methods":"","reagents":"","patternDescription":"As the most abundant biological lifeforms on the planet, bacteriophages provide critical roles within microbes and help regulate their populations (Hatfull 2015). Characterizing newly isolated bacteriophages expands not only our knowledge, but it may also reveal novel genes within their genomes that hold biological significance for our species (Bailly-Bechet et al. 2007). This knowledge can provide insight into the ever-expanding host-virus dynamic and theoretically give rise to therapeutic clinical applications, for example to isolate and evaluate bacteria with pathogenic specificity to combat potential diseases (Pope et al. 2015). As antibiotic resistance continues to rise, phage therapy has gained renewed interest as a potential alternative treatment. Due to their high specificity and ability to lyse bacterial cells, bacteriophages may offer a promising solution to multidrug-resistant infections (Zalewska-Piątek 2023).
The Microbacterium phage BaronJohn was isolated from a soil sample collected in Brunswick, GA (31°10'56\" N, 81° 21' 59\" W) using an enriched isolation protocol (Zorawik et al. 2024). The soil sample was incubated in a peptone-yeast-calcium (PYCa) liquid medium at 32°C while shaking for two hours at 360 RPM using a Labnet Orbit™ 300 Digital Multipurpose Shaker. After incubation, the sample was centrifuged at 2,000 x g for 10 minutes and the supernatant was filtered through a 0.22 µm pore size. This supernatant was collected in a vented 50 ml conical tube, seeded with 0.5 ml of Microbacterium foliorum, and incubated at 32°C with shaking at 360 RPM for three days. Following incubation, the supernatant was filtered through a 0.22 µm pore size membrane, and serial dilutions (10-1 to 10-8 µl) were plated with host M. foliorum on PYCa medium with soft agar overlay. After 48 hours of incubation at 32°C, clear, well-defined plaques were observed. BaronJohn was purified through four rounds of plaque assays.
Phage DNA was extracted from high-titer lysate (6 x 1010 pfu/ml). Briefly, bacterial DNA and RNA in the lysate was first degraded by treatment with DNase I and RNaseA. Nucleases were subsequently inactivated with EDTA, Proteinase K, and SDS, after which phage DNA was extracted using the Promega Wizard DNA Cleanup System. Libraries were prepped using the NEB II Ultra Library kit and sequenced using Illumina NextSeq 1000, producing 2,501,030 single-end 100-bp reads (5077-fold coverage). Raw reads were trimmed with Cutadapt 4.7 (Miller 2011), using the option: –nextseq-trim 30, and filtered with Skewer 0.2.2 (Jiang et al. 2014), using the options: -q 20 -Q 30 -n -l 50, prior to assembly. BaronJohn's genomic termini were assessed using Consed v29 (Gordon and Green 2013) following Russell (2018) and raw reads were assembled using Newbler v2.9 (Miller et al. 2010). The genome of BaronJohn is 40,502 base pairs long and circularly permuted with a G+C content of 63.9%.
Glimmer v3.02 (Delcher et al. 2007) and GeneMark.hmm v3.36 (Besemer and Borodovsky 2005) were used in the initial auto-annotation to identify regions with coding potential. DNA Master v5.23.6 (Pope and Jacobs-Sera 2017) and Starterator v587 (http://phages.wustl.edu/starterator/) were used to review these predictions and revise as needed. BLASTp v2.2.26 (Altschul et al. 1990) with the Actinobacteriophage and NCBI non-redundant databases and PhagesDB (Russell and Hatfull 2016) were used to compare sequences and functions of similar genes. Phamerator, using Actino-draft database v643 (Cresawn et al. 2011) was used to compare gene location, length, and conserved domains among closely related phages. HHpred MPI Bioinformatics Toolkit with databases PDB_mmCIF70_20_Feb, SCOPe70_2.08, Pfam-A_v38.2, NCBI_Conserved_Domains (CD)_v3.19, and UniProt-SwissProt-viral70_3_Nov_2021 was used to analyze protein crystalline structure to predict function (Söding et al. 2005). DeepTMHMM v1.044 (Chen et al. 2003) was used to identify transmembrane proteins. Aragorn v1.2.41 was used to confirm the presence and location of tRNA genes (Laslett and Canback 2004). Default settings were used for all software.
BaronJohn was assigned to subcluster EA5 due to at least 35% shared gene content similarity to phages in the Actinobacteriophage database (https://phagesDB.org; Russell and Hatfull 2016; Pope et al. 2017). The G+C content of 63.9% falls within the range (63.5-64.4%) for subcluster EA5 phages. BaronJohnhas a total of 61 putative genes, 24 of which were assigned functions, and one tRNA-Ala gene (see Fig. 1 for details). This is consistent with the 15 other phages in subcluster EA5 to date, whose number of genes range from 57-63 per genome, and 10 of 15 phages which also have a tRNA gene that is either Ala or undetermined, and syntenic (https://phagesDB.org). Five of the genes in BaronJohn were found to contain transmembrane domains, although the function could not be determined. Roughly half of the genes (28) are transcribed in the forward direction, with the other half (33) transcribed from the opposite strand. Like all other EA5 phages, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a MazG-like nucleotide pyrophosphohydrolase, and two glycosyltransferases (all EA5 phages annotated to date have either one or two in tandem). All 16 EA5 phages encode a lysin A (previously called endolysin) downstream of the minor tail proteins, although these genes comprise two different gene families (Phamilies). No genes canonically associated with lysogeny (e.g. integrase, immunity repressor) were identified on this genome, suggesting a virulent (lytic) life cycle.
Nucleotide Accession Number
GenBank Accession Number is PZ575424. The Sequence Read Archive (SRA) number is SRX32199476.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research 17: 1486-1495.
","pubmedId":"","doi":"10.1101/gr.6649807"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.
","pubmedId":"","doi":"10.1128/JVI.01340-15"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. In M. Clokie, A. Kropinski, & R. Lavigne (Eds.), Bacteriophages (Vol. 1681, pp. 109–125). Humana Press.
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zalewska-Piątek B. 2023. Phage Therapy—Challenges, Opportunities and Future Prospects. Pharmaceuticals 16: 1638.
","pubmedId":"","doi":" 10.3390/ph16121638"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence of Actinobacteriophage BaronJohn, Isolated on Microbacterium foliorum on the College of Coastal Georgia Campus
","reviews":[],"curatorReviews":[]},{"id":"77b1f580-e42a-4c02-807b-f470d9826ab4","decision":"publish","abstract":"Actinobacteriophage BaronJohn was isolated from soil collected near the Lakeside dormitory building on the campus of the College of Coastal Georgia using Microbacterium foliorum NRRL B-24224 as a host. BaronJohn has a genome length and gene content similar to that of phages in cluster EA5. Specifically, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a tRNA-Ala gene, two glycosyltransferases in tandem and a lysin A downstream of the minor tail proteins.
","acknowledgements":"We thank the SEA-PHAGES program and Howard Hughes Medical Institute for supporting this research. We thank Graham Hatfull, Deborah Jacobs-Sera, Daniel Russell, Rebecca Garlena and Colleen Knight for their technical support. We thank the SEA MINT team for their thorough review of this manuscript.
","authors":[{"affiliations":["University of Georgia, Athens, Georgia USA"],"departments":["College of Public Health"],"credit":["writing_reviewEditing","investigation"],"email":"John.Barron@uga.edu","firstName":"John","lastName":"Barron","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920156676@student.ccga.edu","firstName":"John","lastName":"Barton","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920055698@student.ccga.edu","firstName":"Brittni","lastName":"Brown","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920149231@student.ccga.edu","firstName":"Madeleine","lastName":"Culbreath","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920147718@student.ccga.edu","firstName":"Phoebe Gerlyn","lastName":"De Jesus ","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920137401@student.ccga.edu","firstName":"Elena","lastName":"Doppel","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920158442@student.ccga.edu","firstName":"Paige","lastName":"Gailey","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920143290@student.ccga.edu","firstName":"Brenda","lastName":"Garcia","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920149421@student.ccga.edu","firstName":"Mark","lastName":"Quarterman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920164802@student.ccga.edu","firstName":"Kylie","lastName":"Thomas","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920148457@student.ccga.edu","firstName":"Joanna","lastName":"Thomason","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920116221@student.ccga.edu","firstName":"Felicia","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["investigation","writing_originalDraft"],"email":"920141727@student.ccga.edu","firstName":"Kevin","lastName":"Tran","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":""},{"affiliations":["College of Coastal Georgia, Brunswick, Georgia USA"],"departments":["Natural Sciences"],"credit":["supervision","validation","investigation","visualization","writing_reviewEditing","writing_originalDraft"],"email":"hnance@ccga.edu","firstName":"Holly","lastName":"Nance","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":true,"WBId":null,"orcid":""}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"We thank the College of Coastal Georgia Department of Natural Sciences for supporting this work.
","image":{"url":"https://portal.micropublication.org/uploads/bcc90766848cb3a782f59606c5a39f59.png"},"imageCaption":"Genes are presented as boxes along a genome ruler (in kilobases), with different colors representing different gene families based on sequence similarity. Boxes above the ruler represent genes that are transcribed in the forward direction and genes below the ruler are transcribed in the reverse direction. All predicted gene functions are labeled.
","imageTitle":"Phamerator map of BaronJohn’s genome
","methods":"","reagents":"","patternDescription":"As the most abundant biological lifeforms on the planet, bacteriophages provide critical roles within microbes and help regulate their populations (Hatfull 2015). Characterizing newly isolated bacteriophages expands not only our knowledge, but it may also reveal novel genes within their genomes that hold biological significance for our species (Bailly-Bechet et al. 2007). This knowledge can provide insight into the ever-expanding host-virus dynamic and theoretically give rise to therapeutic clinical applications, for example to isolate and evaluate bacteria with pathogenic specificity to combat potential diseases (Pope et al. 2015). As antibiotic resistance continues to rise, phage therapy has gained renewed interest as a potential alternative treatment. Due to their high specificity and ability to lyse bacterial cells, bacteriophages may offer a promising solution to multidrug-resistant infections (Zalewska-Piątek 2023).
The Microbacterium phage BaronJohn was isolated from a soil sample collected in Brunswick, GA (31°10'56\" N, 81° 21' 59\" W) using an enriched isolation protocol (Zorawik et al. 2024). The soil sample was incubated in a peptone-yeast-calcium (PYCa) liquid medium at 32°C while shaking for two hours at 360 RPM using a Labnet Orbit™ 300 Digital Multipurpose Shaker. After incubation, the sample was centrifuged at 2,000 x g for 10 minutes and the supernatant was filtered through a 0.22 µm pore size. This supernatant was collected in a vented 50 ml conical tube, seeded with 0.5 ml of Microbacterium foliorum, and incubated at 32°C with shaking at 360 RPM for three days. Following incubation, the supernatant was filtered through a 0.22 µm pore size membrane, and serial dilutions (10-1 to 10-8 µl) were plated with host M. foliorum on PYCa medium with soft agar overlay. After 48 hours of incubation at 32°C, clear, well-defined plaques were observed. BaronJohn was purified through four rounds of plaque assays.
Phage DNA was extracted from high-titer lysate (6 x 1010 pfu/ml). Briefly, bacterial DNA and RNA in the lysate was first degraded by treatment with DNase I and RNaseA. Nucleases were subsequently inactivated with EDTA, Proteinase K, and SDS, after which phage DNA was extracted using the Promega Wizard DNA Cleanup System. Libraries were prepped using the NEB II Ultra Library kit and sequenced using Illumina NextSeq 1000, producing 2,501,030 single-end 100-bp reads (5077-fold coverage). Raw reads were trimmed with Cutadapt 4.7 (Miller 2011), using the option: –nextseq-trim 30, and filtered with Skewer 0.2.2 (Jiang et al. 2014), using the options: -q 20 -Q 30 -n -l 50, prior to assembly. BaronJohn's genomic termini were assessed using Consed v29 (Gordon and Green 2013) following Russell (2018) and raw reads were assembled using Newbler v2.9 (Miller et al. 2010). The genome of BaronJohn is 40,502 base pairs long and circularly permuted with a G+C content of 63.9%.
Glimmer v3.02 (Delcher et al. 2007) and GeneMark.hmm v3.36 (Besemer and Borodovsky 2005) were used in the initial auto-annotation to identify regions with coding potential. DNA Master v5.23.6 (Pope and Jacobs-Sera 2017) and Starterator v587 (http://phages.wustl.edu/starterator/) were used to review these predictions and revise as needed. BLASTp v2.2.26 (Altschul et al. 1990) with the Actinobacteriophage and NCBI non-redundant databases and PhagesDB (Russell and Hatfull 2016) were used to compare sequences and functions of similar genes. Phamerator, using Actino-draft database v643 (Cresawn et al. 2011) was used to compare gene location, length, and conserved domains among closely related phages. HHpred MPI Bioinformatics Toolkit with databases PDB_mmCIF70_20_Feb, SCOPe70_2.08, Pfam-A_v38.2, NCBI_Conserved_Domains (CD)_v3.19, and UniProt-SwissProt-viral70_3_Nov_2021 was used to analyze protein crystalline structure to predict function (Söding et al. 2005). DeepTMHMM v1.044 (Chen et al. 2003) was used to identify transmembrane proteins. Aragorn v1.2.41 was used to confirm the presence and location of tRNA genes (Laslett and Canback 2004). Default settings were used for all software.
BaronJohn was assigned to subcluster EA5 due to at least 35% shared gene content similarity to phages in the Actinobacteriophage database (https://phagesDB.org; Russell and Hatfull 2016; Pope et al. 2017). The G+C content of 63.9% falls within the range (63.5-64.4%) for subcluster EA5 phages. BaronJohnhas a total of 61 putative genes, 24 of which were assigned functions, and one tRNA-Ala gene (see Fig. 1 for details). This is consistent with the 15 other phages in subcluster EA5 to date, whose number of genes range from 57-63 per genome, and 10 of 15 phages which also have a tRNA gene that is either Ala or undetermined, and syntenic (https://phagesDB.org). Five of the genes in BaronJohn were found to contain transmembrane domains, although the function could not be determined. Roughly half of the genes (28) are transcribed in the forward direction, with the other half (33) transcribed from the opposite strand. Like all other EA5 phages, BaronJohn encodes a tail assembly chaperone with a -1 translational frameshift, a MazG-like nucleotide pyrophosphohydrolase, and two glycosyltransferases (all EA5 phages annotated to date have either one or two in tandem). All 16 EA5 phages encode a lysin A (previously called endolysin) downstream of the minor tail proteins, although these genes comprise two different gene families (Phamilies). No genes canonically associated with lysogeny (e.g. integrase, immunity repressor) were identified on this genome, suggesting a virulent (lytic) life cycle.
Nucleotide Accession Number
GenBank Accession Number is PZ575424. The Sequence Read Archive (SRA) number is SRX32199476.
","references":[{"reference":"Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403-410.
","pubmedId":"","doi":"10.1016/S0022-2836(05)80360-2"},{"reference":"Bailly-Bechet M, Vergassola M, Rocha E. 2007. Causes for the intriguing presence of tRNAs in phages. Genome Research 17: 1486-1495.
","pubmedId":"","doi":"10.1101/gr.6649807"},{"reference":"Besemer J, Borodovsky M. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33: W451-W454.
","pubmedId":"","doi":"10.1093/nar/gki487"},{"reference":"Chen Y, Yu P, Luo J, Jiang Y. 2003. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mammalian Genome 14: 859-865.
","pubmedId":"","doi":"10.1007/s00335-003-2296-6"},{"reference":"Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12: 10.1186/1471-2105-12-395.
","pubmedId":"","doi":"10.1186/1471-2105-12-395"},{"reference":"Delcher AL, Bratke KA, Powers EC, Salzberg SL. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679.
","pubmedId":"","doi":"10.1093/bioinformatics/btm009"},{"reference":"Gordon D, Green P. 2013. Consed: a graphical editor for next-generation sequencing. Bioinformatics 29: 2936-2937.
","pubmedId":"","doi":"10.1093/bioinformatics/btt515"},{"reference":"Hatfull GF. 2015. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. Journal of Virology 89: 8107-8110.
","pubmedId":"","doi":"10.1128/JVI.01340-15"},{"reference":"Jiang H, Lei R, Ding SW, Zhu S. 2014. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15: 10.1186/1471-2105-15-182.
","pubmedId":"","doi":"10.1186/1471-2105-15-182"},{"reference":"Laslett D. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 32: 11-16.
","pubmedId":"","doi":"10.1093/nar/gkh152"},{"reference":"Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10.
","pubmedId":"","doi":"10.14806/ej.17.1.200"},{"reference":"Miller JR, Koren S, Sutton G. 2010. Assembly algorithms for next-generation sequencing data. Genomics 95: 315-327.
","pubmedId":"","doi":"10.1016/j.ygeno.2010.03.001"},{"reference":"Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, et al., undefined. 2015. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife 4: 10.7554/elife.06416.
","pubmedId":"","doi":"10.7554/eLife.06416"},{"reference":"Pope WH, Jacobs-Sera D. 2017. Annotation of Bacteriophage Genome Sequences Using DNA Master: An Overview. Methods in Molecular Biology,Bacteriophages : 217-229.
","pubmedId":"","doi":"10.1007/978-1-4939-7343-9_16"},{"reference":"Pope WH, Mavrich TN, Garlena RA, Guerrero-Bustamante CA, Jacobs-Sera D, Montgomery MT, et al., Hatfull. 2017. Bacteriophages of\n Gordonia\n spp. Display a Spectrum of Diversity and Genetic Relationships. mBio 8: 10.1128/mbio.01069-17.
","pubmedId":"","doi":"10.1128/mbio.01069-17"},{"reference":"Russell DA, 2018. Sequencing, assembling, and finishing complete bacteriophage genomes. In M. Clokie, A. Kropinski, & R. Lavigne (Eds.), Bacteriophages (Vol. 1681, pp. 109–125). Humana Press.
","pubmedId":"","doi":""},{"reference":"Russell DA, Hatfull GF. 2016. PhagesDB: the actinobacteriophage database. Bioinformatics 33: 784-786.
","pubmedId":"","doi":"10.1093/bioinformatics/btw711"},{"reference":"Soding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Research 33: W244-W248.
","pubmedId":"","doi":"10.1093/nar/gki408"},{"reference":"Zalewska-Piątek B. 2023. Phage Therapy—Challenges, Opportunities and Future Prospects. Pharmaceuticals 16: 1638.
","pubmedId":"","doi":" 10.3390/ph16121638"},{"reference":"Zorawik M, Jacobs-Sera D, Freise AC, SEA-PHAGES, Reddi K. 2024. Isolation of Bacteriophages on Actinobacteria Hosts. Methods in Molecular Biology,Phage Engineering and Analysis : 273-298.
","pubmedId":"","doi":"10.1007/978-1-0716-3798-2_17"}],"title":"Genome Sequence of Actinobacteriophage BaronJohn, Isolated on Microbacterium foliorum on the College of Coastal Georgia Campus
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