Mycobacteriophage Faze9 is a novel lytic phage that was isolated from soil collected in Scranton, PA, using the host Mycobacterium smegmatis mc2155. Faze9 has a double-stranded DNA genome composed of 67,503 kbp, has a G+C content of 68.9%, encodes 91 predicted proteins, and is closely related to mycobacteriophages in the B2 subcluster.
ABSTRACT
Mycobacteriophage Faze9 is a novel lytic phage that was isolated from soil collected in Scranton, PA, using the host Mycobacterium smegmatis mc2155. Faze9 has a double-stranded DNA genome composed of 67,503 kbp, has a G+C content of 68.9%, encodes 91 predicted proteins, and is closely related to mycobacteriophages in the B2 subcluster.
ANNOUNCEMENT
Bacteriophage Faze9 was isolated from the host organism Mycobacterium smegmatis mc2155. M. smegmatis mc2155 is a Gram-positive, acid-fast, rod-shaped aerobic bacterium. M. smegmatis is a nonpathogenic bacterium that serves as the model system for the analysis of Mycobacterium tuberculosis genes (1). Faze9 was isolated from soil collected in Scranton, PA (41.4319N, 75.6322W), using an enrichment protocol (2) and the bacterial host M. smegmatis mc2155, by the Marywood University phage hunters as part of the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program of the Howard Hughes Medical Institute (3). Plaques were clear and approximately 1 mm in diameter.
Faze9 is a member of actinobacteriophage subcluster B2. Based on morphological analysis using electron microscopy, Faze9 exhibits a Siphoviridae morphotype with an icosahedral head diameter of approximately 48 nm and a tail length of 283 nm. Genomic DNA was isolated from Faze9 lysate with a Wizard DNA extraction kit (Promega), following the kit protocol. A sequencing library was prepared from genomic DNA using a NEBNext Ultra II FS kit with dual-indexed barcoding and was run on an Illumina MiSeq system, which yielded ∼920,000 single-end 150-base reads, representing ∼1,962-fold coverage of the genome. The raw reads were assembled using Newbler 2.9, with default settings, and yielded a single phage contig, which was checked for completeness, accuracy, and phage genomic termini using Consed 29 and methods described previously (4). Faze9 contains a circularly permuted genome consisting of 67,503 kbp, with 68.9% G+C content and a predicted start codon distribution of 66% ATG and 34% GTG. The G+C contents of the host bacterium (M. smegmatis mc2155) and Faze9 were similar, being 67.4% and 68.9%, respectively (5). Genome annotation was performed manually after the completion of preliminary annotation by DNA Master 5.23.2 (http://cobamide2.bio.pitt.edu). Protein-coding gene prediction was executed by using Glimmer 3.02 (6) and GeneMark 2.5p (7), and functional assignments were made by employing NCBI BLASTP with the Conserved Domain Database at NCBI (8), Actinobacteriophage Database BLAST (https://phagesdb.org/blast), HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred) (9), and Phamerator (https://phamerator.org) (10). Annotations predicted 91 open reading frames with 24 known functional genes (24.6% of the genome); 5.1% of the Faze9 genome represented noncoding nucleotides. No tRNA genes were identified.
This genome contains genes with predicted functions related to phage “DNA” packaging (HNH endonuclease, head-to-tail adaptor, portal protein, and terminase), gene and virion structure (minor tail protein and tape measure protein), DNA replication (RuvC-like resolvase, DNA helicase, and DNA primase/polymerase), host lysis (lysin A and holin), and DNA modifications (ParB-like nuclease domain protein, queine tRNA-ribosyltransferase, QueC, QueD, and Que-like queosine (Genebank term) biosynthesis protein), as well as 67 hypothetical proteins. Faze9 shows genome-wide similarity to the other 11 lytic phages in this cluster, sharing 99.34% identity with its closest relative, Coffee (GenBank accession number MK620898). Comparison to Coffee was performed through a nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov). The subcluster B2 bacteriophages Arbiter, Hedgerow, and LizLemon each had 99% similarity to Faze9, with 100% coverage (NCBI BLAST).
Data availability.
The complete genome sequence of Faze9 was deposited in GenBank with accession number MK061411, BioProject accession number PRJNA488469, and SRA accession number SRX6867480.
ACKNOWLEDGMENTS
We thank the Science, Math, and Computer Science Department of Marywood University and the Howard Hughes Medical Institute SEA-PHAGES program for their support. We thank Craig Johnson at Drexel University for electron microscopy image support and Welkin H. Pope and Graham F. Hatfull at Pittsburgh University for their critical review of this announcement, as well as Daniel A. Russell for whole-genome sequencing support.
This research was not financially supported by any grant agency in the public, commercial, or nonprofit sectors. This work was financially supported by Marywood University.
Marywood University SEA-PHAGES 2017 annotators were as follows: Logan Brighton, Angelo Brutico, Mariah Casey, Ama Gajanayake, Christopher S. McCann, Emily E. Medwid, Emily P. Onyshczak, Dekonti E. Sayeh, Toni Shipman, Alyssa R. Tirella, and Julianna Turnbach. Marywood University SEA-PHAGES 2016 hunters were as follows: Logan Brighton, Mariah Casey, Marlee M. Holleran, Christina N. Kazanas, Patricia R. Lee, Christopher S. McCann, Emily E. Medwid, Emma L. Moser, Emily P. Onyshczak, Dekonti E. Sayeh, and Alyssa R. Tirella.
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Associated Data
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Data Availability Statement
The complete genome sequence of Faze9 was deposited in GenBank with accession number MK061411, BioProject accession number PRJNA488469, and SRA accession number SRX6867480.