Here, we report the genome sequence of LuckyBarnes, a newly isolated singleton siphovirus that infects Brevibacterium iodinum ATCC 15728 and has a 50,774-bp genome with 67 predicted genes

D\u27Angelo, William A.

Foto, Amanda

Gurung, Chandan

Hartley, Shelby G.

Kennedy, Keyshawn L.

Mavrodi, Dmitri V.

Nelms, Alexander E.

Pope, Welkin H.

Ray, Amanda F.

Ryals, Logan M.

Underwood, Savannah L.

English

Aquila Digital Community (University of Southern Mississippi, USM)

The University of Southern Mississippi The Aquila Digital Community Faculty Publications 6-20-2019 Discovery and Characterization of Bacteriophage LuckyBarnes Savannah L. Underwood University of Southern Mississippi Amanda Foto University of Southern Mississippi Amanda F. Ray University of Southern Mississippi Alexander E. Nelms University of Southern Mississippi Keyshawn L. Kennedy University of Southern Mississippi See next page for additional authors Follow this and additional works at: https://aquila.usm.edu/fac_pubs  Part of the Biology Commons Recommended Citation Underwood, S. L., Foto, A., Ray, A. F., Nelms, A. E., Kennedy, K. L., Hartley, S. G., Ryals, L. M., Gurung, C., D'Angelo, W. A., Pope, W. H., Mavrodi, D. V. (2019). Discovery and Characterization of Bacteriophage LuckyBarnes. Microbiology Resource Announcements(25), 1-2. Available at: https://aquila.usm.edu/fac_pubs/16424 This Article is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Faculty Publications by an authorized administrator of The Aquila Digital Community. For more information, please contact Joshua.Cromwell@usm.edu. Authors Savannah L. Underwood, Amanda Foto, Amanda F. Ray, Alexander E. Nelms, Keyshawn L. Kennedy, Shelby G. Hartley, Logan M. Ryals, Chandan Gurung, William A. D'Angelo, Welkin H. Pope, and Dmitri V. Mavrodi This article is available at The Aquila Digital Community: https://aquila.usm.edu/fac_pubs/16424 Discovery and Characterization of Bacteriophage LuckyBarnesSavannah L. Underwood,a Amanda Foto,a Amanda F. Ray,a Alexander E. Nelms,a Keyshawn L. Kennedy,a Shelby G. Hartley,aLogan M. Ryals,a Chandan Gurung,a William A. D’Angelo,a* Welkin H. Pope,b Dmitri V. MavrodiaaDepartment of Cell and Molecular Biology, School of Biological, Environmental, and Earth Sciences, The University of Southern Mississippi, Hattiesburg, Mississippi, USAbDepartment of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USAABSTRACT Here, we report the genome sequence of LuckyBarnes, a newly isolatedsingleton siphovirus that infects Brevibacterium iodinum ATCC 15728 and has a50,774-bp genome with 67 predicted genes.Actinobacteria encompass saprophytes, commensals, and pathogens, as well asproducers of anticancer, anthelmintic, and antifungal secondary metabolites (1).Bacteriophages play a crucial role in the evolution of Actinobacteria and provideinsights into the genetics and physiology of this economically important group ofbacteria. Despite the apparent importance of actinobacteriophages, our understandingof their biology is limited to viruses of Mycobacterium, Gordonia, and Arthrobacter, whilethe knowledge of phages that infect other members of Actinobacteria is lagging (2). Wereport here the characterization of a new bacteriophage, LuckyBarnes, which wasisolated from soil collected in D’Iberville, MS, using enrichment with Brevibacteriumiodinum ATCC 15728 in a peptone yeast calcium (PYCa) medium. Genome analysisidentified LuckyBarnes as a singleton siphovirus and as one of the only two Brevibac-terium phages with sequenced genomes in the Actinobacteriophage database (3).The bacteriophage was recovered by passing the enrichment culture through a0.22-m filter and incubating the filtrate with B. iodinum at room temperature for 48 h,which resulted in 1-mm clear plaques. Transmission electron microscopy of Lucky-Barnes revealed a 50-nm capsid and a 250-nm-long flexible tail (Fig. 1). Phage DNA wasisolated using the Wizard DNA cleanup kit (Promega) and used to prepare a library witha NEBNext Ultra II FS kit (New England BioLabs), which was then sequenced on anIllumina MiSeq instrument with MiSeq v3 chemistry. The sequence run generated 1.42million 150-bp single-end reads that were assembled in Newbler v.2.9 (4), with defaultparameters, to give a 50,774-bp contig with 3,954-fold coverage and a GC content of61.9%. No defined genomic termini could be identified, and to preserve gene conti-guity, the genome start point was selected 2.74 kb upstream of the predicted terminasegene.The genome sequence was analyzed with GeneMark v.3.25 (5), GLIMMER (6),tRNAscan-SE v.2.0 (7), and ARAGORN v.1.2.38 (8), followed by manual annotation usingDNA Master v.5.0.2 (http://cobamide2.bio.pitt.edu/computer.htm), Starterator (https://seaphages.org/software/), and HHpred (9). Phamerator (10) analysis of the 67 predictedprotein-coding genes revealed that most of them (86%) lack homologs in otheractinobacteriophages. Several structural genes were similar to their counterparts fromthe Arthrobacter phage TripleJ (https://phagesdb.org/phages/TripleJ/). The genes ac-count for a 94.7% coding capacity of the genome and are arranged into two genomearms transcribed in divergent directions, which is similar to cluster A mycobacterio-phages (11). The 29.6-kb left genome arm contains virion structure and assembly genes,followed by genes for a terminase, a portal protein, a tail terminator, a tape measureprotein, a major tail subunit, minor tail proteins, and lytic enzymes represented by aclass II holin and an N-acetylmuramoyl-L-alanine amidase. Genes within the 20.6-kbCitation Underwood SL, Foto A, Ray AF, NelmsAE, Kennedy KL, Hartley SG, Ryals LM, GurungC, D’Angelo WA, Pope WH, Mavrodi DV. 2019.Discovery and characterization ofbacteriophage LuckyBarnes. Microbiol ResourAnnounc 8:e00330-19. https://doi.org/10.1128/MRA.00330-19.Editor John J. Dennehy, Queens CollegeCopyright © 2019 Underwood et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Dmitri V. Mavrodi,dmitri.mavrodi@usm.edu.* Present address: William A. D’Angelo,McGowan Institute of Regenerative Medicine,University of Pittsburgh, Pittsburgh,Pennsylvania, USA.Received 27 April 2019Accepted 30 May 2019Published 20 June 2019GENOME SEQUENCEScrossmVolume 8 Issue 25 e00330-19 mra.asm.org 1 on July 10, 2019 by guesthttp://mra.asm.org/Downloaded from right genome arm encode several DNA metabolism enzymes, including a DNA poly-merase, a primase, a DNA helicase, a single-stranded (ssDNA) binding protein, as wellas a predicted dCMP-hydroxymethylase and two glycosyltransferases that may modifyits DNA and improve resistance to degradation by host nucleases. The rest of thepredicted genes (42%) encode conserved hypothetical proteins. Clear plaques and theabsence of an integrase gene suggest that LuckyBarnes is a lytic phage.Data availability. The genome of bacteriophage LuckyBarnes was deposited inDDBJ/ENA/GenBank under the accession number MF668275. The raw reads are avail-able in the SRA under accession number SRR8782908.ACKNOWLEDGMENTSWe thank Daniel A. Russell and Rebecca A. Garlena for conducting genome sequenc-ing, quality control, and assembly at the University of Pittsburgh.We acknowledge support from the Mississippi INBRE, funded by an InstitutionalDevelopment Award (IDeA) from the National Institute of General Medical Sciences ofthe National Institutes of Health under grant P20GM103476. This project was alsosupported by The University of Southern Mississippi and the Howard Hughes MedicalInstitute Science Education Alliance-Phage Hunters Advancing Genomics and Evolu-tionary Science program (www.seaphages.org).REFERENCES1. Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Meier-Kolthoff JP, Klenk H-P, Clément C, Ouhdouch Y, van Wezel GP. 2016.Taxonomy, physiology, and natural products of Actinobacteria. Micro-biol Mol Biol Rev 80:1– 43. https://doi.org/10.1128/MMBR.00019-15.2. Hatfull GF. 2018. Mycobacteriophages. Microbiol Spectr 6:GPP3-0026-2018. https://doi.org/10.1128/microbiolspec.GPP3-0026-2018.3. Russell DA, Hatfull GF. 2017. PhagesDB: the actinobacteriophage data-base. Bioinformatics 33:784–786. https://doi.org/10.1093/bioinformatics/btw711.4. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA,Berka J, Braverman MS, Chen Y-J, Chen Z, Dewell SB, Du L, Fierro JM,Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Ho CH, Irzyk GP, JandoSC, Alenquer MLI, Jarvie TP, Jirage KB, Kim J-B, Knight JR, Lanza JR,Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB,McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, PucBP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M,Tartaro KR, Tomasz A, Vogt KA, et al. 2005. Genome sequencing inmicrofabricated high-density picolitre reactors. Nature 437:376 –380.https://doi.org/10.1038/nature03959.5. Besemer J, Borodovsky M. 2005. GeneMark: Web software for genefinding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res 33:W451–W454. https://doi.org/10.1093/nar/gki487.6. Kelley DR, Liu B, Delcher AL, Pop M, Salzberg SL. 2012. Gene predictionwith Glimmer for metagenomic sequences augmented by classificationand clustering. Nucleic Acids Res 40:e9. https://doi.org/10.1093/nar/gkr1067.7. Lowe TM, Chan PP. 2016. tRNAscan-SE on-line: search and contextualanalysis of transfer RNA genes. Nucleic Acids Res 44:W54 –W57. https://doi.org/10.1093/nar/gkw413.8. Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genesand tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16.https://doi.org/10.1093/nar/gkh152.9. Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, GablerF, Söding J, Lupas AN, Alva V. 2018. A completely reimplemented MPIbioinformatics toolkit with a new HHpred server at its core. J Mol Biol430:2237–2243. https://doi.org/10.1016/j.jmb.2017.12.007.10. Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF.2011. Phamerator: a bioinformatic tool for comparative bacteriophagegenomics. BMC Bioinformatics 12:395. https://doi.org/10.1186/1471-2105-12-395.11. Hatfull GF. 2012. The secret life of mycobacteriophages. Adv Virus Res82:179 –288. https://doi.org/10.1016/B978-0-12-394621-8.00015-7.FIG 1 Plaque appearance (left) and virion morphology (right) of bacteriophage LuckyBarnes. Forelectron microscopy, high-titer lysate was applied on Formvar-coated grids, negatively stained with 1%phosphotungstic acid, and imaged with a Zeiss EM-109 transmission electron microscope (Carl Zeiss AG).Underwood et al.Volume 8 Issue 25 e00330-19 mra.asm.org 2 on July 10, 2019 by guesthttp://mra.asm.org/Downloaded from 

Discovery and Characterization of Bacteriophage LuckyBarnes

https://aquila.usm.edu/cgi/viewcontent.cgi?article=17735&context=fac_pubs

Discovery and Characterization of Bacteriophage LuckyBarnes

Abstract

Similar works

Full text

Available Versions

Aquila Digital Community (University of Southern Mississippi, USM)