Mycobacteriophages represent a rich source of viral genomic diversity, and their sequencing supports both fundamental biological insights and educational programs like the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) initiative (Jordan et al., 2014). A deeper understanding of the genetic diversity of bacteriophage genomes may reveal novel genes whose products could have applications in medicine and biotechnology. The genomes of four double-stranded DNA (dsDNA)-tailed bacteriophages (TribleTrouble, TClif, Llorens, and CallaLilly) that infect Mycobacterium smegmatis mc ² 155 are reported here. These phages provide additional representation of cluster K phages and facilitate comparative analyses of genome architecture, gene content, and structural features.

Phages were isolated using standard enrichment techniques (Zorawik et al., 2024). Briefly, soil samples were resuspended in Middlebrook 7H9 liquid medium, inoculated with M. smegmatis mc ² 155 and incubated with shaking at 30˚C. After 48 hours, the cultures were centrifuged, the supernatant was filtered (0.2 μm pore filter) and the filtrate was plated in top agar with M. smegmatis. Plates were incubated at 30˚C for 48 hours. All phages were purified through a minimum of three rounds of plaque assay.

Electron microscopy on negatively stained (uranyl acetate, 1%) particles revealed the phages have siphovirus morphology (Figure 1). Phage genomic DNA was extracted from a lysate using the Promega Wizard Cleanup Kit. Libraries were prepared using the NEB Ultra II FS kit, and sequenced on an Ilumina MiSeq 1000 (TribleTrouble, TClif and Llorens; v3 reagents) or a NextSeq1000 (CallaLilly; XLEAP-P1 kit). The raw reads generated on the MiSeq1000 were assembled with Newbler v2.9 to generate single contigs. The raw reads generated on the NextSeq1000 were trimmed with cutadapt 4.7 (using the option: –nextseq-trim 30) and filtered with skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) prior to assembly (Martin, 2011, Jiang et al., 2014, Wick et al, 2017). The genomes were checked for completeness and termini type using Consed v29 (Gordon and Green, 2013, Russell, 2017). Additional sequencing and genome details are summarized in Table 1.

Genes were predicted using Glimmer v3.02 (Delcher et al., 2007), Genemark.hmm v3.36, GenemarkS v4.28 (Besemer and Borodovsky, 2005), Aragorn v1.2.38 (Laslett and Canback, 2004) and tRNAscan-SE v2.0.12 (Lowe and Eddy, 1997). Manual inspection and revision were performed using PECAAN v20250130 (Rinehart et al, 2016). Within PECAAN, gene functions were assigned using HHPRED (Söding et al., 2005) alignment to the PDB_mmCIF70, Pfam- v.36, NCBI’s Conserved Domains databases and BLAST (Altschul et al., 1990), alignment to the NCBI nonredundant protein (National Center for Biotechnology Information) and PhagesDB (Russell and Hatfull, 2016) databases. Default settings were used for all software. Based on gene content similarity the phages were assigned to specific actinobacteriophage subclusters (Table 1) (Pope et al, 2017; Russell and Hatfull, 2016).

Functions were predicted for approximately 49% of the called genes across all four phages. Structural proteins (e.g. major capsid and tail proteins) are located in the left region of the genomes, followed by the lysis cassette, which includes the lysin A, holin, and lysin B genes. The predicted immunity repressor of all four phages belong to the same pham; a feature shared with phages known to form lysogens. (Cresawn et. al., 2011, Pope et al., 2011). Similarly, the predicted tyrosine integrases of TClif, Llorens and CallaLilly and the cluster K phages known to form lysogens all belong to the same pham. TribleTrouble encodes a predicted tyrosine integrase that belongs to a different pham, but is present in phages previously shown to establish lysogeny (e.g. Fionnbharth). Predicted DNA metabolism and replication genes are located on the right arm of the genome and nearly all of the predicted genes are transcribed in the forward direction. The exceptions include the predicted immunity repressor, a membrane protein, a secreted protein, and a tRNA gene located in the Llorens and CallaLilly genomes. Although phages tend to display high host specificity, members of the K1 subcluster have been shown to infect both fast- and slow-growing mycobacterial hosts (Pope et al., 2011).

Data availability:

GenBank and Sequence Read Archive (SRA) accession numbers are provided in Table 1