Characterization of four cluster A1 Mycobacterium phage genomes, Applejack, Hermia, LilBib, and QTRlifeCrisis

TL;DR

This paper describes four new Mycobacterium phage genomes and their unique genetic features.

Contribution

The study characterizes four new cluster A1 mycobacteriophages with unique enzymatic features.

Findings

Four new siphoviruses (Applejack, Hermia, LilBib, QTRlifeCrisis) were isolated and sequenced.

Applejack and LilBib encode metallophosphatases with inteins.

Applejack contains a DNA methylase with an out-of-frame HNH endonuclease.

Abstract

Four novel cluster A1 siphoviruses, Applejack, Hermia, LilBib, and QTRlifeCrisis, were isolated on Mycobacterium smegmatis. Their complete genome sequences are 49,633–50,325 bp in length and encode 80–91 protein-coding genes. Applejack and LilBib encode metallophosphatases containing inteins, and Applejack encodes a DNA methylase that contains an out-of-frame HNH endonuclease.

Figures

Genome map of cluster A1 Mycobacterium phages Applejack, Hermia, LilBib, and QTRlifeCrisis. The genome coordinates are represented by the ruler in units of kilobase pairs. The colored boxes above and below the ruler represent genes transcribed in the forward and reverse directions, respectively, and gene or gene product (gp) number is indicated within the box. Genes were assigned to a family using Phamerator in the Actino_draft database, and different families are indicated by colors (13). Colored shading between the genomes indicates nucleotide identity with violet of the color spectrum indicating the highest nucleotide identity. Predicted functions are centered above or below forward and reverse transcribed genes, respectively.

Tables

TABLE 1. Phage and genome characteristics

Isolation data	Applejack	Hermia	LilBib	QTRlifeCrisis
Soil GPS coordinates	44.898789 N, 68.666355 W	44.896201 N, 68.672347 W	44.5549 N, 68.4206 W	44.896201 N, 68.672347 W
Soil isolation location	Orono, ME	Orono, ME	Orono, ME	Old Town, ME
Isolation procedure	Enrichment	Enrichment	Direct	Direct
Rounds of plaque assays to purify plaques	3	4	6	5
Plaque morphology	2–7 mm diameter, clear center turbid ring	4 mm diameter, turbid	4 mm diameter, turbid	2–4 mm, clear center turbid ring
Particle morphology and average particle dimensions (± the standard error)
Morphology	Siphoviridae	Siphoviridae	Siphoviridae	Siphoviridae
Tail length (nm)	167 ± 2.03	171 ± 3.11	201 ± 2.26	199 ± 2.12
Capsid diameter (nm)	69.7 ± 0.724	72.3 ± 0.753	69.6 ± 1.01	72.4 ± 0.733
Number of particles measured	5	5	5	5
Sequencing and genome characteristics
Genome length	49,791	49,670	50,325	49,633
GC content	63.5%	63.9%	63.8%	63.7%
Number 250 bp single-end reads	191,866	192,615	191,557	193,004
Fold coverage	708	836	625	928
Number of protein-coding genes	91	84	80	86
Number of tRNAs	0	0	0	0

Full text

ANNOUNCEMENT

Actinobacteriophage are diverse viruses that infect Actinobacteria, including non-pathogenic and pathogenic Mycobacterium species (1–6). Isolating and characterizing novel phages provides insight to phage evolution and opportunities to improve treatments for antibiotic-resistant mycobacterial infections (5, 7). Phages Applejack, Hermia, LilBib, and QTRlifeCrisis were isolated on the host Mycobacterium smegmatis MC^2^155 using direct and enrichment isolation procedures (Table 1). Soil extracts were prepared in 7H9 top agar, incubated for 1–2 h at 30°C and filtered on a 0.22-µM filter. For direct isolations, soil extracts were inoculated into M. smegmatis, incubated for 15 min and plated in 7H9 top agar onto L-agar plates. For enrichment isolations, soil extracts were inoculated with M. smegmatis, incubated for 2 d at 30°C with shaking before filtering onan 0.22-µM filter. Serially diluted extracts were plated with M. smegmatis and 7H9 top agar onto L-agar plates. Plates were incubated for 2daysd at 30°C and screened for plaques. Multiple rounds of standard plaque assays were performed to purify plaques (Table 1) (8). Hermia and LilBib consistently formed 4-mm turbid plaques. QTRlifeCrisis and Applejack plaques vary in size and have clear centers with turbid rings (Table 1). Transmission electron microscopy revealed particle morphologies consistent with Siphoviridae (Table 1).

Phage genomic DNA was isolated from high-titer lysates by phenol chloroform extraction (9) and prepared for sequencing using the Kapa Hyper Plus DNA library kit (Roche, South San Francisco, CA). Genomes were sequenced on an Illumina NovaSeq 6000 platform, and fold coverage is reported in Table 1. Newbler v.2.9 and Consed v29 were used to assemble genomes and check for completeness (10–12). The four genomes were 49,633–50,325 bp in length, have 63% GC content, and genome ends defined by 10 bp (CGGATGGTAA) 3′ overhangs (Table 1). They share >35% gene content with other cluster A mycobacteriophages in the Phamerator database (Actino_Draft v.589), and were assigned to subcluster A1 (2, 13).

Genomes were auto-annotated using Glimmer v.3.02 and GeneMark v.2.5 embedded in DNAMaster v.5.23.6 and PECAAN (https://blog.kbrinsgd.org/) (14, 15). Translational starts were manually determined based on inclusion of coding potential and conservation across homologs using Starterator (http://phages.wustl.edu/starterator/), BLASTP, and GeneMark.hmm output (14, 16). Putative gene functions were predicted using BLASTP and HHpred (16, 17). Transmembrane domains were predicted using Deep TMHMM (18). No tRNAs were detected using Aragorn v.1.2.38 and tRNAScan-SE2.0 (19, 20). The genomes encode 80–91 protein-coding genes (Table 1). Left arms encode forward-transcribed structural and assembly genes (Fig. 1). Right arms encode reverse-transcribed genes including DNA polymerase I, an immunity repressor, and overlapping DNA primases (Fig. 1). Hermia, LilBib, and QTRlifeCrisis encode a serine integrase that is absent in Applejack.

Genome map of cluster A1 Mycobacterium phages Applejack, Hermia, LilBib, and QTRlifeCrisis. The genome coordinates are represented by the ruler in units of kilobase pairs. The colored boxes above and below the ruler represent genes transcribed in the forward and reverse directions, respectively, and gene or gene product (gp) number is indicated within the box. Genes were assigned to a family using Phamerator in the Actino_draft database, and different families are indicated by colors (13). Colored shading between the genomes indicates nucleotide identity with violet of the color spectrum indicating the highest nucleotide identity. Predicted functions are centered above or below forward and reverse transcribed genes, respectively.

All the genomes encode multiple homing endonucleases and metallophosphatases. However, Applejack and LilBib metallophosphatases (gp48 and 51) contain an intein with high probability HHpred alignment to the Pyrococcus furiosis intein PI-Pfu (Fig. 1) (21). This intein includes a HINT domain for protein splicing and a homing endonuclease domain with two LAGLIDADG motifs, components of the active site (21, 22). The function of the intein is unknown but potentially provides a mechanism of post-translational regulation (23). Applejack also has a DNA methylase (gp54) with an out-of-frame HNH endonuclease (gp55). An HNH endonuclease is found upstream of the small subunit terminase in all genomes and likely plays a role in genome packaging (24).