ABSTRACT
Twelve B1 cluster mycobacteriophages were isolated from soil samples collected in Philadelphia, PA, USA, using Mycobacterium smegmatis mc2 155 as a host, and were sequenced. The genome sequences range in size from 66,887 bp to 68,953 bp in length and have between 99 and 105 putative protein-coding genes.
ANNOUNCEMENT
Mycobacteriophages are viruses that infect and replicate within mycobacteria. Based on their genome sequences, mycobacteriophages have been classified into 29 clusters. The B cluster is the second-most abundant cluster, with 286 members (phagesdb.org). This cluster has been subdivided into 8 subclusters (B1 to B8). In this paper we describe 12 B1 cluster mycobacteriophages isolated and annotated at Drexel University, Philadelphia, PA, USA.
All 12 bacteriophages were isolated from soil samples collected in Philadelphia, PA, USA. The bacterial host used was Mycobacterium smegmatis mc2 155. Phage Gareth was isolated by adding 7H9 liquid medium to the soil sample. The liquid was filtered using a 0.22-µm filter and then incubated with Mycobacterium smegmatis mc2 155 at 37°C for 48 h. The other 11 bacteriophages were isolated by adding 7H9 broth with glycerol, albumin dextrose (AD) supplement, CaCl2, and Mycobacterium smegmatis mc2 155 to the soil samples. They were shaken at 220 rpm at 37°C for 96 h. The samples were centrifuged, and the supernatant was removed and filtered with a 0.22-µm filter. Plaque assays were performed to confirm the isolation of the bacteriophages. The bacteriophages were plaque-purified and amplified, then double-stranded DNA (dsDNA) was extracted using the Promega Wizard DNA cleanup kit. The genomes were individually sequenced with the Illumina MiSeq platform by the Pittsburgh Bacteriophage Institute. Sequencing libraries were prepared from genomic DNA with a NEB Ultra II kit or an Illumina TruSeq Nano kit. Forty-eight libraries were multiplexed and sequenced with an Illumina MiSeq platform, yielding at least 100,000 150-base single-end reads for each genome sequence, which was sufficient to provide at least 150-fold coverage (see Table 1). Raw reads were assembled with Newbler 2.9 with default settings, each yielding a single-phage contig, which was checked for completeness, accuracy, and phage genomic termini with Consed 29. After sequencing, the genomes were annotated with the following databases and software: DNA Master 5.23.2 (http://cobamide2.bio.pitt.edu), Glimmer 3.02 (1), GeneMark 2.5p (2), Starterator, Phamerator (https://phamerator.org) (3), PhagesDB BLAST (phagesdb.org/blast), NCBI BLASTP, the NCBI Conserved Domain Database (4, 5), HHPRED 3.0 beta (6), Aragorn 1.2.38 (7), tRNAscanSE 2.0 (8), and PECAAN (http://pecaan.kbrinsgd.org). The genomes were screened for repeated motifs with MEME software 5.0.2 (9).
Twelve newly isolated B1 cluster mycobacteriophages
The genomic features of these bacteriophages are summarized in Table 1. The genome sequence sizes range from 66,887 to 68,953 bp and have a GC content from 66.3% to 66.5%. Similar to other B1 cluster bacteriophages (10), they contain ∼100 coding sequences (CDS) and no tRNA genes in any of the genomes.
Of these bacteriophage sequences, Phergie and PhenghisKhan are the most similar to each other, with 99% identity over 100% coverage (NCBI BLAST), and the most diverse are PhenghisKhan and Vaticameos, which share 98% identity over 93% coverage (NCBI BLAST). The genomic regions exhibiting the highest level of nucleotide diversity occurred between nucleotide positions 46 kb and 58 kb. Differences between the bacteriophage sequences include an HNH endonuclease gene (gp10) present in JangoPhett, Kailash, PhrodoBaggins, Phunky, and Virapocalypse, similar to that in phage Murdoc (GenBank accession number JN638752). Gareth and Vaticameos have a truncated version of the gene. Repeated motifs were identified; for example, one 50 nucleotide motif shared by bacteriophages Phunky, Gareth, Virapocalypse, JangoPhett, and Vaticameos was identified 42 nucleotides upstream of a shared forward gene, which has no known function.
Data availability.The genome sequences reported here have been deposited in DDBJ/ENA/GenBank under the accession numbers provided in Table 1 (BioProject number PRJNA488469). The SRA/DRA/ERA accession numbers are also provided in Table 1.
ACKNOWLEDGMENTS
This research was conducted as part of the Science Education Alliance (SEA) Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program, supported by the Howard Hughes Medical Institute (HHMI). This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
We thank Graham F. Hatfull, Deborah Jacobs-Sera, Daniel A. Russell, and Rebecca A. Garlena for their technical support during the sequencing and annotation of this genome.
The contributing authors were Shreeya R. Agrawal, Katielee Cummings, Nishanth R. Eluri, Viktor S. Evtimov, Anuranita Gupta, Ali H. Harb, Devneet K. Kainth, Saira Khan, Benjamin D. Koa, Danny T. Kuriakose, Elizabeth S. McCluskey, Arjun S. Menon, Sina Mortazavi, Sharon G. Nam, Tyler D. Pettigrew, Cat-Thi A. Phan, Felicia M. Raju, Amber N. Richardson, Christian N. Schill, Danielle M. Senk, Shayna P. Singh, Sharmayne T. Siu, Natalia Sivchuk, Carly J. Smith, Sonia S. Varandani, and Guna Yerrabolu (Drexel University SEA-PHAGES annotators 2016), Lavanya S. Aluri, Dharman Anandarajan, Jamison C. Beiriger, Raviteja Bethamcharla, Niteesha Betini, Shivangi D. Bhatt, Nicole E. Cox, Bela P. Delvadia, Aishwary S. Desai, Andrew M. Devaney, Brenna K. Doyle, Arden O. Edgerton, Kevin C. Fitzpatrick, Esha A. Gajjar, Anjali Ganguly, Ramnik S. Gill, Madeleine G. Goldman, Pauline M. Good, Nishtha Gupta, Leila M. Haddad, Esther J. Han, Shelby Jain, Andrew Jiang, Andrew D. Jurgielewicz, Jawhara M. Karam, Mallika Kodavatiganti, Sinja J. Kriete, Catherine E. MacDonald, Sravani Meka, Josh P. Maret, Ashley E. Mathew, Nusrat M. Nishu, Nirali Patel, Pooja D. Patel, Shivani Patel, Kaustav Patra, Daechaton Pumpuckdee, Karima K. Rai, Arghyadeep Sarkar, Brianna L. Schaffer, Priyanka Shah, Andrew H. Tawfik, Bhavya T. Thuremella, Justina Toma, Shika Veera, Vamsee K. Vemulapalli, Trevor V. Vidas, Katy S. Vieira, Gayathri Vijayakumar, Clara R. White, and Shu l. Zhao (Drexel University SEA-PHAGES annotators 2017), and Akash M. Bhat, Ishaan J. Bhatt, Praneetha S. Bheemarasetty, Madison R. Bockol, Zachary Cataline, Upoma Chakraborty, Amanda C. Chen, Nina H. Cheng, Hannah Cherusseril, Cara DePiano, Leah Dobossy, Skylar Driscoll, Jinie Eom, Rahul Gandhi, Ashley L. Harkins, Hannah S. Johnson, Shraddha V. Kamat, Un Bi Kim, Victoria G. Koa, Zhuo Kuang, Hannah J. Lee, Angelo R. Lodato, Richard Lu, Shreya Mandloi, Rohil Mediratta, Sreya Muchivolu, Tania H. Mulherkar, Emily Navarreto, Chandler J. Olson, Chanyeong Park, Riya Patel, Dhwanil J. Patel, Uyen X. H. Phan, Apollonia Quiros, Mitali M. Shah, Saloni Shah, Shannon Smith, Allison C. Tran, Alyssa Truxon, Kanyinsola Yoloye, Nari Yoon, and Nathan D. Zhang (Drexel University SEA-PHAGES annotators 2018).
FOOTNOTES
- Received 12 November 2018.
- Accepted 18 January 2019.
- Published 21 February 2019.
- Copyright © 2019 Apellido et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.