Abstract
Mycobacteriophages are viruses that infect mycobacterial hosts. The current collection of sequenced mycobacteriophages—all isolated on a single host strain, Mycobacterium smegmatis mc2155, reveals substantial genetic diversity. The complete genome sequences of 63 newly isolated mycobacteriophages expand the resolution of our understanding of phage diversity.
GENOME ANNOUNCEMENT
The bacteriophage population encompasses amazing genetic diversity, reflecting its vast size, dynamic nature, and ancient origins (1–4). The collection of sequenced mycobacteriophage genomes offers insights into phages that infect a common host, Mycobacterium smegmatis mc2155 (5, 6), and thus are presumed to be in genetic communication with one another. Comparative genomics reveals mosaicism as the fundamental architectural feature of phage genomes, which are constructed from segments exchanged among the phage population (7, 8). Nonetheless, simple DNA comparison identifies groups of genomes more similar to one another than to other phages, and these are referred to as clusters (9). The previously reported 223 mycobacteriophages assort into a total of 15 different clusters (clusters A through O) and eight singletons that have no close relatives (5, 10–13). Several of the clusters can be divided into subclusters according to nucleotide sequence similarities (5). The total number of clusters, subclusters, and singletons is 36 (5).
Bacteriophage discovery and genomic characterization provide an effective platform for introducing students to authentic scientific research (9, 14, 15). The 63 phages described here were isolated and sequenced by undergraduate students in four programs: the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) Program, the Phage Hunters Integrating Research and Education (PHIRE) Program, the Research Immersion Laboratory in Virology at UCLA, and the Mycobacterial Genetics Course at the University of KwaZulu-Natal, Durban, South Africa (Table 1). Students and faculty isolated, purified, named, extracted, and annotated each of the new phages.
TABLE 1.
Data for newly sequenced mycobacteriophage genomes
| Phage name | Cluster | Accession no. | Length (bp) | GC% | Institution(s) |
|---|---|---|---|---|---|
| ABCat | E | KF188414 | 76,131 | 63.0 | Southern Connecticut State Universitya |
| Aeneas | A1 | JQ809703 | 53,684 | 63.6 | Brigham Young Universitya |
| ArcherS7 | C1 | KC748970 | 156,558 | 64.7 | Trinity Collegea |
| Arturo | A4 | JX307702 | 51,500 | 64.1 | Hampden-Sydney Collegea |
| Astraea | C1 | KC691257 | 154,872 | 64.7 | North Carolina State Universitya |
| Astro | A8 | JX015524.1 | 52,494 | 61.4 | College of Charlestona |
| Ava3 | C1 | JQ911768 | 154,466 | 64.8 | Calvin Collegea |
| Avani | F2 | JQ809702 | 54,470 | 61.0 | Gettysburg Collegea |
| Bobi | F1 | KF114874 | 59,179 | 61.7 | Purdue Universitya |
| Breeniome | C1 | KF006817 | 154,434 | 64.8 | CUNY, Queens Collegea |
| Breezona | L2 | KC691254 | 76,652 | 58.9 | Bucknell Universitya |
| Butters | N | KC576783 | 41,491 | 65.8 | Lehigh Universitya |
| Catdawg | O | KF017002 | 72,108 | 65.4 | Cabrini Collegea |
| Contagion | E | KF024732 | 74,533 | 63.1 | Loyola Marymount Universitya |
| Crossroads | L2 | KF024731 | 76,129 | 58.9 | Teacher workshop, University of Pittsburghd |
| Daenerys | F1 | KF017005 | 58,043 | 61.6 | University of Pittsburgha |
| Dhanush | A4 | KC661271 | 51,373 | 63.9 | University of Alabama at Birminghama |
| Dorothy | F1 | JX411620 | 58,866 | 61.4 | Baylor Universitya |
| Dumbo | E | KC691255 | 75,736 | 63.0 | University of Maine, Fort Kenta |
| Dylan | O | KF024730 | 69,815 | 65.4 | University of KwaZulu-Natalb |
| ElTiger69 | A5 | JX042578 | 51,505 | 59.8 | University of North Texasa |
| Fishburne | P | KC691256 | 47,109 | 67.3 | College of Charlestona |
| Flux | A4 | JQ809701 | 51,370 | 63.9 | Saint Joseph’s Universitya |
| Gizmo | C1 | KC748968 | 157,482 | 64.6 | Illinois Wesleyan Universitya |
| Goose | A10 | JX307704 | 50,645 | 65.1 | Del Mar Collegea |
| Hamulus | F1 | KF024723 | 57,155 | 61.8 | University of California, Santa Cruza |
| HINdeR | A7 | KC661275 | 52,617 | 62.8 | Miami Universitya |
| ICleared | A4 | JQ896627 | 51,440 | 63.9 | University of Louisiana at Monroea |
| Jabbawokkie | F2 | KF017003 | 55,213 | 61.1 | University of Pittsburgha |
| Job42 | F1 | KC661280 | 59,626 | 61.2 | Providence Collegea |
| Jobu08 | A3 | KC661281 | 50,679 | 64.0 | Washington State Universitya |
| KayaCho | B4 | KF024729 | 70,838 | 70.0 | University of KwaZulu-Natala,b |
| LittleCherry | A5 | KF017001 | 50,690 | 60.9 | College of William & Marya |
| MacnCheese | K3 | JX042579 | 61,567 | 67.3 | Calvin Collegea |
| Marcell | A1 | JX307705 | 49,186 | 64.0 | University of Maine, Machiasa |
| Medusa | A4 | KF024733 | 51,384 | 63.9 | University of Louisiana at Monroea |
| Methuselah | A3 | KC661272 | 50,891 | 64.2 | University of Maryland, Baltimore Countya |
| Muddy | Single | KF024728 | 48,228 | 58.8 | University of KwaZulu-Natalb |
| Murphy | E | KC748971 | 76,179 | 62.9 | Ohio State Universitya |
| Newman | B1 | KC691258 | 68,598 | 66.5 | University of Colorado at Bouldera |
| Odin | A2 | KF017927 | 52,807 | 62.3 | University of California, Los Angelesc |
| PattyP | A1 | KC661273 | 52,057 | 63.6 | Carthage Collegea |
| PegLeg | M | KC900379 | 80,955 | 61.5 | University of California, Los Angelesc |
| Phaux | E | KC748969 | 76,479 | 62.9 | Helena High School,d Queensboro Community Collegea |
| Phrux | E | KC661277 | 74,711 | 63.1 | Miami Universitya |
| Rebeuca | A10 | JX411619 | 51,235 | 65.1 | University of Texas at El Pasoa |
| Redno2 | J | KF114875 | 108,297 | 60.9 | Virginia Commonwealth Universitya |
| Reprobate | B5 | KF024727 | 70,120 | 68.3 | University of KwaZulu-Natalb |
| Sabertooth | A4 | JX307703 | 51,377 | 63.9 | Culver-Stockton Collegea |
| SarFire | A1 | KF024726 | 53,701 | 63.8 | University of KwaZulu-Natalb |
| SDcharge11 | B1 | KC661274 | 67,702 | 66.5 | Loyola Marymount Universitya |
| Severus | A10 | KC661279 | 49,894 | 64.4 | College of St. Scholasticaa |
| ShiVal | B1 | KC576784 | 68,355 | 66.5 | Montclair State Universitya |
| Shrimp | C1 | KF024734 | 155,714 | 64.7 | Illinois Wesleyan Universitya |
| SiSi | F1 | KC661278 | 56,279 | 61.5 | Gonzaga Universitya |
| Taj | F1 | JX121091 | 58,550 | 61.9 | University of Pittsburghd |
| Tiger | A5 | JQ684677 | 50,332 | 60.7 | Oregon State Universitya |
| Trouble | A1 | KF024724 | 52,102 | 63.6 | University of California, Santa Cruza |
| Twister | A10 | JQ512844 | 51,094 | 65.0 | Virginia Commonwealth Universitya |
| Velveteen | F1 | KF017004 | 54,314 | 61.5 | University of Pittsburgha |
| Wanda | J | KF006818 | 109,960 | 60.8 | Lehigh Universitya |
| Whirlwind | L3 | KF024725 | 76,050 | 59.3 | University of Pittsburghd |
| Winky | L2 | KC661276 | 76,653 | 58.9 | Saint Joseph’s Universitya |
SEA-PHAGES member institution.
K-RITH Mycobacterial Genetics Course.
UCLA Research Immersion Laboratory in Virology.
Phage Hunters Integrating Research and Education (PHIRE) Program, University of Pittsburgh.
Phage genomic DNA was sequenced using Ion Torrent, 454 pyrosequencing, or Illumina platforms with an average redundancy of 210-fold (range, 25- to 900-fold). Reads were assembled using Newbler (version 2.6), and the assembly quality was evaluated using Consed (version 20 or newer); targeted Sanger sequencing resolved weak areas where needed. Fifty-two of the genomes have defined ends, and the termini were determined by comparison to closely related genomes, examination of read density, and where necessary, Sanger sequencing on genomic DNA. Eleven genomes assembled circularly and are presumed to have circularly permuted terminally redundant ends, and position 1 was selected at the 5′ end of the most distally closely linked gene to the terminase large subunit (1 to 5 genes upstream in the five cluster B phages). For the six cluster C phages, position 1 was chosen for consistency with Bxz1 (8).
Phage genomes were annotated using DNA Master (http://cobamide2.bio.pitt.edu/computer.htm), Glimmer 3.0 (16), GeneMark (17, 18), Aragorn (19), tRNAscan-SE (20), and Phamerator (21), and gene functions were predicted using BLAST and HHPred (22). All sequences and student-annotated genomes were reviewed and revised as necessary at the University of Pittsburgh.
Of the 63 genomes, one (Fishburne) joins a singleton to form cluster P, one (Muddy) is a new singleton, and four add new subclusters, two in A10 and one each in B5 and L5 (Table 1). The 285 sequenced mycobacteriophage genomes form a total of 39 clusters or subclusters and 8 singletons.
Further information on these phage genomes is available at http://www.phagesdb.org.
Nucleotide sequence accession numbers.
GenBank accession numbers are shown in Table 1.
ACKNOWLEDGMENTS
We thank the many undergraduates and instructors who contributed to this phage discovery and genomics initiative. We also thank David J. Asai, Cheryl Bailey, Lucia P. Barker, William R. Bishai, Charles Bowman, Kevin W. Bradley, Darisa Clarke, Nell Eisenberg, Roger Hendrix, William R. Jacobs, Jr., Deborah Jacobs-Sera, Paras Jain, Victoria Kaspowicz, Michelle H. Larsen, Welkin H. Pope, Eric Rubin, and Daniel A. Russell for their assistance in phage isolation, genome analysis, and program administration. Phage genomes were sequenced at the Genomics and Proteomics Core Laboratories and at the Pittsburgh Bacteriophage Institute at the University of Pittsburgh, the David H. Murdock Research Institute, the Nucleic Acids Research Facilities at Virginia Commonwealth University, the Purdue University Genomics Core Facility, the University of California—Santa Cruz’s Genome Sequencing Center, and the UCLA Genotyping and Sequencing Core Facility.
This work was supported by Public Health Service grant GM93901 from the Institute of General Medical Sciences and by the Howard Hughes Medical Institute, including grant 52006944 to UCLA.
Footnotes
Citation Hatfull GF, Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Program, KwaZulu-Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course, University of California—Los Angeles Research Immersion Laboratory in Virology, Phage Hunters Integrating Research Education Program. 2013. Complete genome sequences of 63 mycobacteriophages. Genome Announc. 1(6):e00847-13. doi:10.1128/genomeA.00847-13.
REFERENCES
- 1. Hatfull GF, Hendrix RW. 2011. Bacteriophages and their genomes. Curr. Opin. Virol. 1:298–303 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Hendrix RW. 2002. Bacteriophages: evolution of the majority. Theor. Popul. Biol. 61:471–480 [DOI] [PubMed] [Google Scholar]
- 3. Suttle CA. 2007. Marine viruses—major players in the global ecosystem. Nat. Rev. Microbiol. 5:801–812 [DOI] [PubMed] [Google Scholar]
- 4. Wommack KE, Colwell RR. 2000. Virioplankton: viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 64:69–114 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Hatfull GF, Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Program. KwaZulu-Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course Students. Phage Hunters Integrating Research and Education Program 2012. Complete genome sequences of 138 mycobacteriophages. J. Virol. 86:2382–2384 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Hatfull GF. 2012. The secret lives of mycobacteriophages. Adv. Virus Res. 82:179–288 [DOI] [PubMed] [Google Scholar]
- 7. Hendrix RW, Smith MC, Burns RN, Ford ME, Hatfull GF. 1999. Evolutionary relationships among diverse bacteriophages and prophages: all the world’s a phage. Proc. Natl. Acad. Sci. U. S. A. 96:2192–2197 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Pedulla ML, Ford ME, Houtz JM, Karthikeyan T, Wadsworth C, Lewis JA, Jacobs-Sera D, Falbo J, Gross J, Pannunzio NR, Brucker W, Kumar V, Kandasamy J, Keenan L, Bardarov S, Kriakov J, Lawrence JG, Jacobs WR, Hendrix RW, Hatfull GF. 2003. Origins of highly mosaic mycobacteriophage genomes. Cell 113:171–182 [DOI] [PubMed] [Google Scholar]
- 9. Hatfull GF, Pedulla ML, Jacobs-Sera D, Cichon PM, Foley A, Ford ME, Gonda RM, Houtz JM, Hryckowian AJ, Kelchner VA, Namburi S, Pajcini KV, Popovich MG, Schleicher DT, Simanek BZ, Smith AL, Zdanowicz GM, Kumar V, Peebles CL, Jacobs WR, Jr, Lawrence JG, Hendrix RW. 2006. Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet. 2:e92. 10.1371/journal.pgen.0020092 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Stella EJ, Franceschelli JJ, Tasselli SE, Morbidoni HR. 2013. Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria. PLoS One 8:e56384. 10.1371/journal.pone.0056384 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Pope WH, Jacobs-Sera D, Best AA, Broussard GW, Connerly PL, Dedrick RM, Kremer TA, Offner S, Ogiefo AH, Pizzorno MC, Rockenbach K, Russell DA, Stowe EL, Stukey J, Thibault SA, Conway JF, Hendrix RW, Hatfull GF. 2013. Cluster J mycobacteriophages: intron splicing in capsid and tail genes. PLoS One 8:e69273. 10.1371/journal.pone.0069273 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Fan X, Teng T, Wang H, Xie J. 2012. Biology of a novel mycobacteriophage, SWU1, isolated from Chinese soil as revealed by genomic characteristics. J. Virol. 86:10230–10231 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Mageeney C, Pope WH, Harrison M, Moran D, Cross T, Jacobs-Sera D, Hendrix RW, Dunbar D, Hatfull GF. 2012. Mycobacteriophage Marvin: a new singleton phage with an unusual genome organization. J. Virol. 86:4762–4775 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hanauer DI, Jacobs-Sera D, Pedulla ML, Cresawn SG, Hendrix RW, Hatfull GF. 2006. Inquiry learning. Teaching scientific inquiry. Science 314:1880–1881 [DOI] [PubMed] [Google Scholar]
- 15. Hatfull GF. 2010. Bacteriophage research: gateway to learning science. Microbe Mag. 5:243–250 [Google Scholar]
- 16. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27:4636–4641 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Borodovsky M, Lomsadze A. 2011. Gene identification in prokaryotic genomes, phages, metagenomes, and EST sequences with GeneMarkS suite. Curr. Protoc. Bioinformatics 35:4.5.1–4.5.17. 10.1002/0471250953.bi0405s35 [DOI] [PubMed] [Google Scholar]
- 18. Borodovsky M, Mills R, Besemer J, Lomsadze A. 2003. Prokaryotic gene prediction using GeneMark and GeneMark.hmm. Curr. Protoc. Bioinformatics 1:4.5.1–4.5.16. 10.1002/0471250953.bi0405s01 [DOI] [PubMed] [Google Scholar]
- 19. Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 32:11–16 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955–964 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfull GF. 2011. Phamerator: a bioinformatic tool for comparative bacteriophage genomics. BMC Bioinformatics 12:395 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Söding J, Biegert A, Lupas AN. 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33:W244–W248 [DOI] [PMC free article] [PubMed] [Google Scholar]