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. 2012 Feb;86(4):2382–2384. doi: 10.1128/JVI.06870-11

Complete Genome Sequences of 138 Mycobacteriophages

Graham F Hatfull a,✉; the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Programb; the KwaZulu-Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course Studentsc; the Phage Hunters Integrating Research and Education Programd
PMCID: PMC3302410  PMID: 22282335

Abstract

Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages—the viruses of mycobacterial hosts—provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which—together with the 83 mycobacteriophages previously reported—represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc2 155.

GENOME ANNOUNCEMENT

The bacteriophage population is large (9), dynamic (8), and old (3). The genetic diversity of the population is sufficiently great that phages of different bacterial hosts typically share little recognizable sequence similarity (2). Comparing the genomes of phages that infect a single common bacterial host—and are therefore likely to be in genetic contact with one another—provides insights into both viral diversity and the mechanisms giving rise to new viruses (4). An emerging central theme is that phage genomes are pervasively mosaic, with segments being readily exchanged between genomes by horizontal exchange and thus present in numerous genomic contexts with different genetic neighbors (2). Eighty-three complete mycobacteriophage genome sequences have been previously reported (5, 6) and are grouped into 22 clusters or subclusters except for 5 singletons that have no close relatives (5, 6).

The isolation, purification, and genomic analysis of new bacteriophages offer an effective platform for providing authentic research experiences to novice scientists (1). Of the 138 phages whose genomes are reported here, 80 were isolated by participants in the Howard Hughes Medical Institute Science Education Alliance's Phage Hunters Advancing Genomics and Evolutionary Science Program (SEA-PHAGES), 10 were isolated in a mycobacterial genetics course at the University of KwaZulu-Natal, and 47 were isolated by teachers, undergraduates, and high school students in the Phage Hunters Integrating Research and Education Program (PHIRE) at the University of Pittsburgh. Each of the new phages was plaque purified, named, sequenced, and annotated by participating students and faculty. In addition, the previously isolated phage DS6A—the only one of the 138 phages that does not infect M. smegmatis (7)—was sequenced and annotated.

Genomic DNA isolated from each phage was shotgun sequenced using either Sanger sequencing, 454 pyrosequencing (6), or Illumina sequencing. The qualities of the sequence assemblies were evaluated, and targeted Sanger sequencing was used to resolve weak areas and to determine genome ends. All student-annotated genomes were reviewed and revised as necessary at the University of Pittsburgh prior to GenBank submission.

Of the 138 new genomes, 19 do not belong within an existing cluster and form five new clusters (J, L, M, N, and O), incorporating 3 prior singletons to form new clusters J, L, and O. Six new singletons were identified. Representatives of all existing clusters with the exception of cluster H were identified, and several clusters were further divided to generate subclusters A5 to A9, B5, K4, and K5; cluster L is divided into L1 and L2. The entire collection of 221 genomes forms a total of 36 clusters or subclusters and 8 singletons.

Although the number of mycobacteriophage genomes that have been completely sequenced has grown substantially, the discovery of new clusters, subclusters, and singletons suggests that the population of these phages remains largely undersampled.

Further information on these phage genomes is available at http://www.phagesdb.org. Subsequent reports will describe genomic details and comparative analyses of the phages announced herein.

Nucleotide sequence accession numbers.

The sequences of the 138 newly determined mycobacteriophage genomes have been deposited in GenBank under the accession numbers listed in Table 1.

Table 1.

Newly sequenced mycobacteriophage genomes

Phage Clustera GenBank accession no. Length (bp) Institutionb
ABU B1 JF704091 68,850 1Lehigh U
Acadian B5 JN699007 69,864 2U Pitt
Airmid A5 JN083853 51,241 1UT El Paso
Akoma B3 JN699006 68,711 2U Pitt
Alice C1 JF704092 153,401 1U N Texas
Alma A9 JN699005 53,177 2U Pitt
Arbiter B2 JN618996 67,169 1U Mary Wash
Ares B2 JN699004 67,436 2U Pitt
Athena B3 JN699003 69,409 2U Pitt
Avrafan G JN699002 41,901 2U Pitt
Babsiella I1 JN699001 48,420 2U Pitt
Backyardigan A4 JF704093 51,308 1W Kentucky U
BAKA J JF937090 111,688 2U Pitt
BarrelRoll K1 JN643714 59,672 1W Kentucky U
Bask21 E JF937091 74,997 2U Pitt
Benedict A5 JN083852 51,083 1Baylor U
BigNuz Single JN412591 48,984 3UKZN
BillKnuckles A1 JN699000 51,821 2U Pitt
Blue7 A6 JN698999 52,288 2U Pitt
Bongo M JN699628 80,228 1WUSL
BPBiebs31 A1 JF957057 53,171 1St Joseph's U
Bruns A1 JN698998 53,003 2U Pitt
Charlie N JN256079 43,036 1VCU
ChrisnMich B4 JF704094 70,428 1Culver-Stockton
Courthouse J JN698997 110,569 2IU Southeast
Cuco A5 JN408459 50,965 1UPR Cayey
Daisy B3 JF704095 68,245 1St Joseph's U
Dandelion C1 JN412588 157,568 3UKZN
DaVinci A6 JF937092 51,547 2U Pitt
DeadP F1 JN698996 56,461 2U Pitt
DLane F1 JF937093 58,899 2U Pitt
Doom A1 JN153085 51,421 1Coll William & Mary
Dori Single JN698995 64,613 1UCSC
DotProduct F1 JN859129 55,363 1UCSD
Drago F1 JN542517 54,411 1JMU
Drazdys C1 JF704116 156,281 1UMBC
Dreamboat A1 JN660814 51,083 1Lehigh U
DS6A Single JN698994 60,588 N/A
Elph10 E JN391441 74,675 1UW River Falls
EricB A6 JN049605 51,702 1NC Cen U; 1QCC
Euphoria A1 JN153086 53,597 1Lehigh U
Eureka E JN412590 76,174 3UKZN
Faith1 L2 JF744988 75,960 1Spelman
Fionnbharth K4 JN831653 58,076 1UCSD
Firecracker O JN698993 71,341 1UCSC
Gadjet B3 JN698992 67,949 2U Pitt
George A5 JF704107 51,578 1JMU
Ghost C1 JF704096 155,167 1Cabrini Coll
Gladiator A6 JF704097 52,213 1WUSL
GUmbie F1 JN398368 57,387 1Gonzaga
Hammer A6 JF937094 51,889 2U Pitt
Harvey B1 JF937095 68,193 2U Pitt
Hedgerow B2 JN698991 67,451 2U Pitt
HelDan A3 JF957058 50,364 1Loyola M'Mount
Henry E JF937096 76,049 2U Pitt
Hertubise B1 JF937097 68,675 2U Pitt
Ibhubesi F1 JF937098 55,600 3UKZN
IsaacEli B1 JN698990 68,839 2U Pitt
JacAttac B1 JN698989 68,311 2U Pitt
JAWS K1 JN185608 59,749 1UL Monroe
JC27 A1 JF937099 52,169 2U Pitt
Jeffabunny A6 JN699019 48,963 1UCSC
JHC117 A3 JF704098 50,877 1U Col Boulder
JoeDirt L1 JF704108 74,914 1CUNY Queens
Kamiyu B3 JN699018 68,633 2U Pitt
Kikipoo B1 JN699017 68,839 2U Pitt
KLucky39 B1 JF704099 68,138 1BYU
KSSJEB A1 JF937110 51,381 2U Pitt
Kugel A1 JN699016 52,379 2U Pitt
Larva K5 JN243855 62,991 1Coll William & Mary
Lesedi A1 JF937100 50,486 3UKZN
LHTSCC A4 JN699015 51,813 2U Pitt
Liefie G JN412593 41,650 3UKZN
Lilac E JN382248 76,260 1UPR Cayey
LinStu C1 JN412592 153,882 3UKZN
LittleE J JF937101 109,086 2U Pitt
Marvin Single JF704100 65,100 1Cabrini Coll
MeeZee A4 JN243856 51,368 1CUNY Queens
Microwolf A3 JF704101 50,864 1NC State
MoMoMixon C1 JN699626 154,573 1Jacksonville St
Morgushi B1 JN638753 68,307 1WUSL
Mozy F1 JF937102 57,278 2U Pitt; 3UKZN
MrGordo A1 JN020140 50,988 1Purdue U
Murdoc B1 JN638752 68,600 1WUSL
Museum A1 JF937104 51,426 2U Pitt
Mutaforma13 F1 JN020142 57,701 1NC State
Nappy C1 JN699627 156,646 3UKZN; 1UT El Paso
Nova D JN699014 65,108 2U Pitt
Oline B1 JN192463 68,720 1CMU
Oosterbaan B1 JF704109 68,735 1Calvin Coll
Optimus J JF957059 109,270 1Hope Coll
OSmaximus B1 JN006064 69,118 1U Alabama Birm
PackMan A9 JF704110 51,339 1UL Monroe
Patience Single JN412589 70,506 3UKZN
Perseus A1 JN572689 53,142 1U Col Boulder
Phipps B1 JF704102 68,293 1CMU; 1Georgia St
Pio C1 JN699013 156,758 2Olin Coll Engin
Pleione C1 JN624850 155,586 1WUSL
Rakim E JN006062 75,706 1UL Monroe
Redi N JN624851 42,594 1WUSL
Rey M JF937105 83,724 2U Pitt
RidgeCB A1 JN398369 50,844 1U Montana
Rockstar A3 JF704111 47,780 1UL Monroe
RockyHorror F1 JF704117 56,719 1UCSC
Rumpelstiltskin L2 JN680858 69,279 1JMU
Saintus A8 JN831654 49,228 1Georgia St
Sebata C1 JN204348 155,286 3UKZN; 2U Pitt
Send513 Single JF704112 71,547 1Georgia St;1CMU
Serendipity B1 JN006063 68,804 1Calvin Coll
SG4 F1 JN699012 59,016 2U Pitt
Shaka A4 JF792674 51,369 1Culver-Stockton; 1Loyola M'Mount
Shauna1 F1 JN020141 59,315 1BYU
ShiLan F1 JN020143 59,794 1VCU
SirDuracell E JF937106 75,793 2U Pitt
SirHarley D JF937107 64,791 2U Pitt
Spartacus F1 JQ300538 61,164 2U Pitt
Stinger B4 JN699011 69,641 2U Pitt
Switzer A1 JF937108 52,298 2U Pitt
TallGrassMM B1 JN699010 68,133 2U Pitt
Thibault J JN201525 106,327 1Bucknell U
Thora B1 JF957056 68,839 1Calvin Coll
ThreeOh3D2 B1 JN699009 68,992 2U Pitt
Timshel A7 JF957060 53,278 1U North Texas
TiroTheta9 A4 JN561150 51,367 1W Kentucky U
Toto E JN006061 75,933 1Cabrini Coll
Trixie A2 JN408461 53,526 1VCU
Turbido A2 JN408460 53,169 1VCU; 1U Montana
UPIE L1 JF704113 73,784 1Oregon State U
Violet A1 JN687951 52,481 1UCSC
Vista B1 JN699008 68,494 2U Pitt
Vix A3 JF704114 50,963 1Hope Coll
Vortex B1 JF704103 68,346 1WUSL
Wally C1 JN699625 155,299 1VCU
Wee F1 HQ728524 59,320 2U Pitt
Wile A4 JN243857 51,308 1VCU
Yoshand B1 JF937109 68,719 2U Pitt
Yoshi F2 JF704115 58,714 1UL Monroe
Zemanar B4 JF704104 71,092 1U Mary Wash
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a

Six genomes could not be grouped with any cluster and are described as singletons (Single).

b

Abbreviations: Baylor U, Baylor University; Bucknell U, Bucknell University; BYU, Brigham Young University; Cabrini Coll, Cabrini College; Calvin Coll, Calvin College; CMU, Carnegie Mellon University; Coll William & Mary, College of William and Mary; Culver-Stockton, Culver-Stockton College; CUNY Queens, City University of New York Queens College; Georgia St, Georgia State University; Gonzaga, Gonzaga University; Hope Coll, Hope College; IU Southeast, Indiana University Southeast; Jacksonville St, Jacksonville State University; JMU, James Madison University; Lehigh U, Lehigh University; Loyola M'Mount, Loyola Marymount University; NC Cen U, North Carolina Central University; NC State, North Carolina State University; Olin Coll Engin, Olin College of Engineering; Oregon St U, Oregon State University; Purdue U, Purdue University; QCC, Queensborough Community College; Spelman, Spelman College; St Joseph's U, Saint Joseph's University; U Alabama Birm, University of Alabama at Birmingham; U Col Boulder, University of Colorado—Boulder; UCSC, University of California, Santa Cruz; UCSD, University of California, San Diego; UL Monroe, University of Louisiana at Monroe; UKZN, University of KwaZulu-Natal; U Mary Wash, University of Mary Washington; UMBC, University of Maryland, Baltimore County; U Montana, University of Montana; U N Texas, University of North Texas; U Pitt, University of Pittsburgh; UPR Cayey, University of Puerto Rico at Cayey; UT El Paso, University of Texas at El Paso; UW River Falls, University of Wisconsin—River Falls; VCU, Virginia Commonwealth University; W Kentucky U, Western Kentucky University; WUSL, Washington University in St. Louis; N/A, not applicable (previously isolated). A superscript “1” designates institutions participating in SEA-PHAGES, a superscript “2” the PHIRE program at the University of Pittsburgh, and a superscript “3” the K-RITH mycobacterial genetics course at the University of KwaZulu-Natal.

ACKNOWLEDGMENTS

We recognize the outstanding contributions of the more than 1,600 undergraduate and high school students who participated in this phage discovery and genomics initiative, as well as those of their teachers and instructors. We thank David J. Asai, Lucia P. Barker, William R. Bishai, Charles Bowman, Kevin W. Bradley, Nell Eisenberg, Roger Hendrix, William R. Jacobs, Jr., Deborah Jacobs-Sera, Paras Jain, Tuajuanda C. Jordan, Victoria Kaspowicz, Razi Khaja, Michelle H. Larsen, Melvina Lewis, Prashini Moodie, Welkin H. Pope, Eric Rubin, Daniel A. Russell, and Willem Sturm for their critical 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 Joint Genome Institute, the Nucleic Acids Research Facilities at Virginia Commonwealth University, the University of California Santa Cruz's Genome Sequencing Center, and the Genome Institute at Washington University in St. Louis.

Funding was provided by NIH grants GM093901 and GM51975 and by the Howard Hughes Medical Institute.

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