Complete genome sequences and characteristics of mycobacteriophages Diminimus, Dulcita, Glaske16, and Koreni

ABSTRACT

Complete genome sequences of four novel mycobacteriophages, Diminimus, Dulcita, Glaske16, and Koreni, isolated from soil are presented. All these bacteriophages belong to subcluster M1, except Koreni that belongs to subcluster A4. Moreover, all have siphovirus morphologies, with genome sizes ranging from 51,055 to 81,156 bp.

ANNOUNCEMENT

Bacteriophages are obligate intracellular parasitic viruses of bacteria (1, 2). These viruses play crucial roles in the global ecosystem and impact not only bacterial physiology, diversity, abundance, and virulence but also human, animal, and plant health (3, 4). On the practical side, bacteriophages have multiple applications, including disease diagnosis, phage therapy, food safety, disinfection, correcting dysbiosis, pest control, biosensing, and bioremediation (4 – 9). Here, we report on four novel lysogenic bacteriophages.

All bacteriophages were isolated from soil samples collected around LeTourneau University in Longview, Texas, in August 2022 (Table 1), using standard protocols (10). Briefly, soil samples were mixed with Middlebrook 7H9 broth prior to spinning (2,000 × g at 4°C) and supernatant filtration (0.22 µm pore size). Filtrates were inoculated with Mycobacterium smegmatis mc²155 cells and incubated at 37°C for 4 days with shaking at 210 rpm, then filtered again. The samples were then plated with M. smegmatis in 7H9 top agar and purified through three 48-h rounds of plating at 37°C. Plaque morphologies were clear (Table 1). Negative-stain transmission electron microscopy showed the four bacteriophages to have a siphovirus morphotype with isometric capsids (diameter, ~60.75 to 68.21 nm) and flexible tails (length, ~131.60 to 333.00 nm; Table 1), measured using ImageJ (11 – 13).

TABLE 1.

Properties of four mycobacteriophages isolated from soil samples collected on August 23, 2022 in Longview, Texas, USA

Data for mycobacterium phage a
Characteristic	Diminimus	Dulcita	Glaske16	Koreni
Soil sampling location GPS coordinates	32.46474 N , 94.7272 W	32.464444 N , 94.7275 W	32.465 N , 94.727778 W	32.463333 N , 94.726389 W
Lysate titer (PFU/mL)	1.8 × 1,010	2.0 × 1,010	4.9 × 109	1.4 × 1,010
Plaque morphology after 48 h at 37°C	Clear with defined edges	Clear with defined edges	Clear with defined edges	Clear with defined edges
Avg plaque diameter (mm [n-value])	0.8 (15)	1.0 (10)	1.7 (3)	1.0 (13)
Approx. shotgun coverage (X)	338	479	360	1,490
Genome size (bp)	80,037	80,038	81,156	51,055
GC content (%)	61.6	61.6	61.6	63.9
Overhang sequence	ACCTCCTGCAA	ACCTCCTGCAA	ACCTCCTGCAA	CGGCCGGTAA
Overhang length (bases)	11	11	11	10
Cluster	M	M	M	A
Subcluster	M1	M1	M1	A4
GenBank accession no.	OR521083	OR553916	OR553909	OR553901
SRA accession no.	SRX19690831	SRX19690832	SRX19690837	SRX19690842
Total no. of reads	185,644	258,101	201,637	528,548
No. of predicted genes	137	137	140	90
No. of predicted tRNAs	19	19	18	0
tRNA type(s)	Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val	Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val	Trp, Asn, Gln, Tyr, Pro, Ser, Phe, Met, Arg, His, Leu, Lys, Gly, Val, Thr, Asp, Glu	–
No. of genes with predicted functions	51	52	54	48
% of genes with predicted functions	37.2	38	38.6	53.3
Key predicted lysogenic life cycle genes	Serine integrase	Serine integrase	Serine integrase	Serine integrase, immunity repressor
No. of orphams	0	0	1	0
Avg capsid size (nm [n-value])	61.53 (10)	60.75 (10)	68.21 (3)	61.56 (10)
Avg tail length (nm [n-value])	323.51 (10)	333.00 (11)	328.30 (3)	131.60 (10)
Isolated by	Faith W Baliraine, Taryn M Pledger, Camila Andrade, Chloe I Wade	Krista L Anderson,Olivia L Donnelly,Hannah L Foree, Kaitlyn E Mathews	Sofia N Mendez, Tadeen Feroz, Amya R Orn, Jerron Hudson	Krista L Anderson,Olivia L Donnelly,Hannah L Foree, Kaitlyn E Mathews

^a All bacteriophages were isolated using the enriched isolation method [reference (10)] and purified through three sequential (37°C, 48 h) rounds of plating with Mycobacterium smegmatis mc²155 cells in Middlebrook 7H9 top agar. Genome sequencing was accomplished using the Illumina Shotgun sequencing method with 150-base single-end reads using the NEB Ultra II Library sequencing kit (v3 reagents). All had 3′ single-stranded overhang genome ends. Genomic termini were identified through buildups of read start positions and variations in genome-wide coverage and manually verified using Consed version 29 [references (14) and (15) ]. All bacteriophages had a siphovirus morphotype and were predicted to be lysogenic based on the presence of predicted lysogeny-related genes.

Genomic DNA was extracted from lysates of titers ranging from 4.9 × 10⁹ to 2 × 10¹⁰ PFU/mL (Table 1) using the Promega Wizard DNA cleanup kit. The NEB Ultra II Library kit was used to prepare the samples for sequencing using Illumina MiSeq (v3 reagents; 150-base single-end reads). Untrimmed reads assembly and verification was performed using Newbler v2.9 (16) and Consed v29 (14, 15). Genome sizes ranged from 51,055 bp (phage Glaske16) to 81,156 bp (phage Koreni) (Table 1). All had 3′ single-stranded overhangs (10–11 bp long) and an average GC content of 62.2% (range: 61.6%–63.9%). This was slightly lower than that of our previous isolates from the same general location and of the isolation host M. smegmatis mc²155 (67.4% GC) (17, 18). Using the gene content similarity (GCS) tool in PhagesDB (19, 20), mycobacteriophages Diminimus, Dulcita, and Glaske16 were assigned to subcluster M1, while Koreni was assigned to subcluster A4 (Table 1) based on ≥35% GCS to other phages in the database.

The genomes were annotated using DNAMaster v5.23.6 (21), Starterator (22), Phamerator (23), BLASTp in NCBI GenBank and PhagesDB (24, 25), GenMark v2.5p (26), HHpred PDB_mmCIF70_17_Apr, Pfam-A_v35, UniProt-SwissProt-viral70_3_Nov_2021 and NCBI_Conserved_Domains_v3.19 databases (27, 28), Glimmer v3.02 (29), DeepTMHMM v. 1.0.24 (30), tRNAscan-SE v2.0 (31, 32), and ARAGORN v1.2.41 (33). Default program settings were utilized in all cases (34). On average, 126 putative protein-coding genes (range: 90–140) and 14 tRNAs (range: 0–19) were predicted (Table 1). Functions were predictable only for 37%–53% of the putative genes across the bacteriophages (Table 1). All bacteriophages had at least one lysogenic lifecycle-associated gene. Diminimus, Dulcita, and Glaske16 encoded serine integrase, while Koreni encoded both serine integrase and an immunity repressor. None had an identifiable gene encoding the excise (Table 1).

DATA AVAILABILITY

Raw reads of all four reported mycobacteriophages are available in the Sequence Read Archive (SRA) database, and their complete genome sequences are available in GenBank database. Their SRA and GenBank accession numbers, together with their respective Uniform Resource Locators, are provided in Table 1. Plaque and TEM images are available in the Actinobacteriophage database and can be accessed by typing the phage name in the database search box. High titer lysates of the phages are archived at the University of Pittsburgh Bacteriophage Institute.