Complete Genome Sequences of 61 Mycobacteriophages.

Mycobacteriophages-viruses of mycobacteria-provide insights into viral diversity and evolution as well as numerous tools for genetic dissection of Mycobacterium tuberculosis Here we report the complete genome sequences of 61 mycobacteriophages newly isolated from environmental samples using Mycobacterium smegmatis mc(2)155 that expand our understanding of phage diversity.

GENOME ANNOUNCEMENT

Bacteriophages are the most numerous biological entities on the planet, with a global population of 1031 particles. With an estimated 1023 productive infections per second worldwide, the population is vast, dynamic, and genetically diverse (1–4). As of March 2016, the National Center for Biotechnology Information (NCBI) lists 1,757 Caudovirales genomes, 318 of which infect Mycobacterium hosts. Previous comparative analyses of mycobacteriophages revealed substantial diversity and mosaic architectures resulting from nonhomologous recombination. Integrated research-education programs such as Phage Hunters Integrating Research and Education (PHIRE) (5), Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) (6), the Mycobacterial Genetics Course at the University of Kwazulu-Natal (K-RITH), and the University of California–Los Angeles’s Research Immersion Laboratory in Virology, isolated, sequenced, and annotated the phages reported here (Table 1) using M. smegmatis as a host.

TABLE 1 

Newly sequenced mycobacteriophage genomes

Phage name	Cluster	Genome (bp)	G+C content (%)	GenBank accession no.	Finding/annotating institution
Alvin	A1	49,577	63.5	KP027205	University of Pittsburgha
ArcherNM	A2	52,561	64.2	KU761559	Washington State University, University of Floridab
Artemis2UCLA	A6	52,344	61.4	KF560333	University of California Los Angelesd
Bactobuster	A2	52,129	63.1	KU568494	North Carolina A&T State Universityb
Bernardo	B3	68,196	67.4	KF493879	University of California–Los Angelesd
Bipper	Y	77,832	67.3	KU728633	University of Pittsburgh, Florida Gulf Coast Universityb
Bricole	M1	81,128	61.6	KT591491	Old Dominion Universityb
Bruin	E	74,210	63.0	KF562099	University of California–Los Angelesd
Brusacoram	P	47,618	67.0	KT347313	College of St. Scholasticab
Carcharodon	N	43,680	66.2	KM588359	Jacksonville State Universityb
Cedasite	G1	41,901	66.6	KT355472	Morehouse Collegeb
Chandler	B3	69,450	67.5	KP027207	University of Pittsburgha
CloudWang3	A6	52,873	61.4	KF560332	University of California–Los Angelesd
Conspiracy	A5	50,755	60.6	KF560330	University of California–Los Angelesd
Cosmo	V	78,229	56.8	KP027195	University of KwaZulu-Natalc
Eidsmoe	A9	52,946	62.5	KU716094	Illinois Wesleyan Universityb
Enkosi	K1	59,052	67.2	KT281789	University of KwaZulu-Natalc
Glass	B2	67,509	69.0	KT880194	Hope Collegeb
Graduation	A1	52,823	63.5	KF560331	University of California–Los Angelesd
HanShotFirst	A1	52,390	63.8	KF493880	University of California–Los Angelesd
HufflyPuff	E	76,323	63.0	KF562100	University of California–Los Angelesd
HyRo	C1	153,714	64.7	KT281790	University of KwaZulu-Natalc
Iracema64	A4	51,637	64.0	KU055616	La Salle Universityb
JAMaL	B4	70,841	68.8	KF493881	University of California–Los Angelesd
JenCasNa	A3	50,877	64.0	KU255188	Howard Hughes Medical Instituteb
Jovo	A5	51,319	60.8	KF493882	University of California–Los Angelesd
Kimberlium	F1	56,826	61.4	KR935214	Gettysburg Collegeb
LadyBird	A2	53,141	63.5	KT588442	St. Edward’s Universityb
Lolly9	L3	75,816	59.3	KT281791	University of KwaZulu-Natalc
Lumos	L3	75,586	59.3	KT372003	Indian River State Collegeb
MichelleMyBell	N	42,240	66.0	KF986246	Nyack Collegeb
Mosby	E	74,533	63.1	KF493883	University of California–Los Angelesd
Nala	E	75,894	63.1	KF562101	University of California–Los Angelesd
NaSiaTalie	A2	52,920	63.4	KU297783	Howard Hughes Medical Instituteb
Numberten	B1	68,607	66.5	KJ194583	University of Pittsburgha
Panchino	N	43,516	65.9	KU935727	Lincoln Universityb
Phamished	B1	68,515	66.5	KR816508	Gettysburg Collegeb
PhatBacter	E	76,217	63.0	KF562102	University of California–Los Angelesd
Phatniss	F1	57,293	61.3	KT279576	Johns Hopkins Universityb
Phrann	N	44,872	66.3	KU935731	Southern Connecticut State Universityb
Pioneer	A9	53,219	62.6	KT285706	Indian River State Collegeb
Pipsqueaks	N	43,679	66.3	KU935730	College of Charlestonb
PopTart	F1	55,094	61.6	KT281792	University of KwaZulu-Natalc
Potter	B1	68,327	66.5	KU867907	University of Kansasb
Romney	A4	51,370	63.9	KU867906	Seton Hill Universityb
Sbash	I2	55,832	65.6	KP027201	University of KwaZulu-Natalc
Seabiscuit	A1	51,781	63.7	KJ194585	University of Pittsburgha
Seagreen	F1	57,766	61.8	KT281793	University of KwaZulu-Natalc
SkinnyPete	N	43,478	66.4	KU935729	Virginia Commonwealth Universityb
Snenia	L3	75,626	59.3	KT281794	University of KwaZulu-Natalc
Sparkdehlily	F1	56,275	61.2	KT895280	James Madison Universityb
Tasp14	A1	51,409	63.9	KT326768	Ohio State Universityb
Texage	A3	50,081	64.0	KT326767	Merrimack Collegeb
TheloniousMonk	A1	52,055	63.6	KT363731	Western Kentucky Universityb
Tres	B2	67,349	68.9	KT365402	James Madison Universityb
Wooldri	A3	50,797	64.0	KT381277	Washington State University, University of Floridab
Xeno	N	42,395	66.8	KU935728	Southern Connecticut State Universityb
Xerxes	N	43,698	66.3	KU935726	University of Floridab
XFactor	F1	55,617	61.7	KT281795	University of KwaZulu-Natalc
Zaka	A6	52,122	61.5	KF560334	University of California–Los Angelesd
Zakhe101	O	69,653	65.5	KT281796	University of KwaZulu-Natalc

Phage Hunters Integrating Research and Education (PHIRE) Program, University of Pittsburgh.

Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES).

K-RITH Mycobacterial Genetics Course.

University of California–Los Angeles, Research Immersion Laboratory in Virology.

Phages were isolated by direct plating of filtered soil extracts or from enriched cultures, followed by plaque purification. Electron microscopy shows that 60 have siphoviral morphotypes, and HyRo is the sole member of the Myoviridae. Most have isometric capsids, the exceptions being Bipper, Sbash, and Zakhe101 with prolate heads. Genomic DNA was extracted from high titer lysates, sheared, and sequenced at the University of Pittsburgh, University of California–Los Angeles, the DOE Joint Genome Institute, or Virginia Commonwealth University using either Sanger, Illumina, Ion Torrent, or 454 technology. Sequence reads were assembled using Newbler (Roche) and Consed (7) and coverage depths range from 47-fold to 2,308-fold, with an average of 200-fold. Sequence assemblies revealed discrete genome ends for 52 phages, and the 9 with circularly permuted assemblies were bioinformatically linearized such that base one was assigned in accord with other mycobacteriophages. Genomes were annotated using DNA Master (http://cobamide2.bio.pitt.edu), Phamerator (8), Glimmer (9), GeneMark (10), Aragorn (11), and tRNAscanSE (12), and functions were determined using the public databases GenBank, Protein DataBase, pfamA, and phagesdb.org with BLAST (13), and HHPred (14). Genomes were assigned to clusters or subclusters as described previously (15).

Notwithstanding the large extant collection of sequenced mycobacteriophage genomes, these newly sequenced phages considerably expand our understanding of mycobacteriophage diversity. Twenty-two are members of the largest cluster, cluster A, but span 7 of the 15 subclusters. The others are broadly distributed across other clusters, including B, C, E, F, G, I, K, L, M, N, O, and P. Cosmo has substantial nucleotide sequence similarity to the singleton phage Wildcat, forming the new cluster V. The eight cluster N phages, Cedasite (G1), and Brusacoram (P) are notable in that they contain integration-dependent immunity systems in which the phage attachment site (attP) is located within the repressor gene (16).

As is typical of other sequenced phage genomes, functions can be assigned to only ~25% of the predicted genes, primarily those involved in virion capsid and assembly and well-conserved genes associated with DNA metabolism. Two of the cluster A genomes (Eidsmoe, ArcherNM) contain partitioning systems in place of integration cassettes; several genomes (e.g., Phrann, Xeno) encode toxin-antitoxin systems; and three encode Lsr2 homologs (Lolly9, Lumos, and Snenia).

Nucleotide sequence accession numbers.

Nucleotide sequence accession numbers for all phages are shown in Table 1.