Complete Genome Sequences of Mycobacteriophages SynergyX, Abinghost, Bananafish, and Delton.

Four lytic mycobacteriophages, namely, SynergyX, Abinghost, Bananafish, and Delton, were isolated from soil in Washington, DC, using the bacterial host Mycobacterium smegmatis mc2155. Analysis of the genomes revealed that they belong to two subclusters of actinobacteriophage cluster B (subclusters B2 and B3) and subcluster D1 of cluster D.

ANNOUNCEMENT

Mycobacteriophages SynergyX, Abinghost, Bananafish, and Delton were isolated from soil samples collected from the campus of Howard University in Washington, DC (SynergyX, 38.9217N, 77.01917W; Abinghost, 38.9206N, 77.01694W; Bananafish, 38.9227N, 77.0208W; Delton, 38.9189N, 77.0183W). Students in the Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program followed the SEA-PHAGES Discovery Guide to isolate viruses with the goal of investigating bacteriophage evolution and diversity (1, 2). The samples were shaken at 37°C in 7H9 broth for 2 h, and a soil filtrate was generated as described previously (3). The filtrate was incubated with Mycobacterium smegmatis mc2155 cells at 37°C for 48 h under aerobic conditions. This enriched sample was filtered (0.22 μm) and plated with M. smegmatis mc2155 cells. Three rounds of purification were conducted for each resulting plaque of interest. Genomic DNA was extracted from high-titer lysates using the Promega Wizard DNA cleanup system (2). An Ultra II FS kit with dual-indexed barcoding (New England Biolabs) was used to generate genomic DNA libraries. Pooled libraries were sequenced using an Illumina MiSeq system to yield 150-base single-end reads (SynergyX, 232,719 reads; Abinghost, 356,927 reads; Bananafish, 382,199 reads; Delton, 264,896 reads). Reads were trimmed and assembled using Newbler v2.9 with default settings, yielding a single contig for each genome. Consed v29 was used to check contigs for completeness and accuracy and to determine phage termini (4–6). The phages had circularly permuted genomes.

The genomes were annotated using the Phage Evidence Collection and Annotation Network (PECAAN) (https://discover.kbrinsgd.org) and DNA Master 5.23.3 (http://cobamide2.bio.pitt.edu/computer.htm). Gene presence, start sites, and putative functions were determined using GLIMMER v3.0, GeneMark v2.5, Starterator v1.1 (https://github.com/SEA-PHAGES/starterator), Phamerator Actino_Draft v402 (https://phamerator.org), BLAST v2.11.0+, PhagesDB, HHpred 3.0, and the NCBI Conserved Domain Database (CDD) v3. ARAGON v1.2.38 and tRNAscan-SE v2.0 were used to detect tRNAs (7–14). PhagesDB and the NCBI SRA Taxonomy Analysis tool were used to assign taxonomy and cluster affiliations based on nucleotide similarity (13, 15). Default parameters were used for all software. Descriptive information for each genome is available in Table 1.

TABLE 1

Genome characteristics and accession numbers for four novel mycobacteriophages

Phage	Sequencing coverage (×)	GenBank accession no.	SRA accession no.	Subcluster	Genome size (bp)	G+C content (%)	No. of protein coding genes	Top BLASTn match (GenBank accession no.)	Identity to top BLASTn match (%)
SynergyX	478	MN444872	SRX10013849	B3	68,980	67.5	104	OrangeOswald (KR080203.1)	99.34
Abinghost	734	MN444873	SRX10013839	B3	68,634	67.6	103	Kamiyu (JN699018.1)	98.25
Bananafish	817	MK814751	SRX10013840	B2	67,366	68.9	93	Calamitous (MZ747518.1)	99.08
Delton	580	MK559427	SRX10013860	D1	64,963	59.6	87	Helpful (MN284908.1)	99.37

Nucleotide similarity suggested that the phages are members of the Siphoviridae family and that three of the phages should be assigned to cluster B, namely, SynergyX (subcluster B3), Abinghost (subcluster B3), and Bananafish (subcluster B2). Delton was affiliated with cluster D (subcluster D1) (16). The genome sizes (64,963 to 68,980 bp) and G+C content values (59.6% to 68.9%) were consistent with those of other members of the same subclusters, based on information available in PhagesDB (13). No tRNAs were detected. The phages were confirmed to be lytic based on the generation of clear plaques upon infection of the host, the presence of lysis system genes, and the absence of lysogeny genes. The lysis system includes lysin A and holin in Bananafish and lysin A and lysin B in SynergyX, Abinghost, and Delton. Also present are genes that encode structural proteins and proteins that play roles in DNA synthesis, modification, packaging, and repair processes.

Data availability.

Complete genome sequences are available in GenBank, and raw sequencing reads are available in the NCBI SRA. Accession numbers are provided in Table 1.