In this study we used laboratory strains of Mycobacterium smegmatis mc(2)155 and Mycobacterium tuberculosis H37Rv acquired from the A.N. Bakh Institute of Biochemistry at the Russian Academy of Science collection, as well as clinical isolates of Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii from the collection of the Federal State Budgetary Institution “NMITs FPI” of the Ministry of Health of the Russian Federation.

The mycobacteria strains were cultivated in Middlebrook 7H9 broth (Himedia, India) with 0.05% Tween-80 (Sigma-Aldrich, USA) and on solid Middlebrook 7H11 agar (Himedia, India), both containing 10% Middlebrook OADC Supplement (HiMedia, India), in a humid atmosphere with 5% CO₂ at 37°C. For M. smegmatis mc(2)155, Tween-80 was not used. In experiments related to phage isolation and cultivation, Middlebrook 7H9 soft agar (0.7%) was used, and CaCl₂ was added to all media at a final concentration of 2 mM for M. smegmatis mc(2)155 and 1 mM for other mycobacteria. All manipulations with bacterial strains were performed under biosafety level 2 and 3 conditions.

The M. smegmatis mc(2)155 strain was used as a host for phage isolation. The isolation of Vic9 phage was carried out as previously described (Gong et al., 2021). Briefly, a soil sample (10 g) was co-incubated with 1 mL of M. smegmatis mc(2)155 suspension grown to mid-log phase and 20 μL of ampicillin (50 mg/mL) in 10 mL MP buffer (50 mM Tris–HCl; 150 mM NaCl; 10 mM MgCl₂; 2 mM CaCl₂; pH 7.5) overnight with agitation. After incubation, the sample was centrifuged at 5,000 g for 10 min at 4°C. The supernatant was first filtered through a 0.45 μm PES membrane (Millipore, USA), and then through a 0.22 μm PES membrane (Millipore, USA). The filtrate (10 mL) was added to 10 mL of double-concentrated TSB (Himedia, India), and inoculated with 2 mL of M. smegmatis mc(2)155 suspension grown to mid-log phase and incubated for 48 h with agitation. The double-layer agar plate method was used to detect and isolate the phage (Kropinski et al., 2009). The resulting lysate was serially diluted in Middlebrook 7H9 broth, then 100 μL aliquots were mixed with soft agar inoculated with M. smegmatis mc(2)155 grown to mid-log phase and poured onto a prewarmed Middlebrook 7H11 agar plate. Three rounds of single plaque purification and re-infection of exponentially growing host strains yielded pure phage suspensions. Subsequently, phage lysates were stored at 4°C.

The phage titer was evaluated by standard spot test methodology (Sarkis and Hatfull, 1998). Serial tenfold dilutions of the phage were spotted in 5 μL drops on double-layer agar containing 100 μL of M. smegmatis mc(2)155 suspension grown to mid-log phase.

The phage lysate was filtered through a 0.22 μm PES membrane (Millipore, USA) and concentrated by ultracentrifugation at 75,000 g for 1 h at 4°C. The phage particles were resuspended in SM buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 8 mM MgSO₄, 0.01% gelatin). A suspension of phages (10¹² plaque-forming units (PFU)/mL) was deposited onto carbon-coated grids (Ted Pella, USA) treated using a glow discharge device K100X (Quorum Technologies Ltd., Lewes, UK). Following one-minute deposition, the suspension was blotted, and the grids were stained with 1% uranyl acetate and subsecuently dried. The images were acquired using a transmission electron microscope (TEM) JEM-1400 (JEOL, Japan) operating at 120 kV.

Mid-log phase M. smegmatis mc(2)155 cells were inoculated in Middlebrook 7H9 broth to a concentration of 1 × 10⁷ CFU/mL. The phage was mixed with bacteria at multiplicities of infection (MOIs) of 0.001, 0.01, 0.1, 1, and 10. After incubation with shaking for 48 h, the phage titer was determined using the spot test method (Sarkis and Hatfull, 1998). The experiment was performed in three biological replicates.

The phage adsorption capacity was determined as previously described (Hendrix and Duda, 1992). Briefly, M. smegmatis mc(2)155 in mid-log phase and Vic9 phage were mixed at a multiplicity of infection (MOI) of 0.01 in Middlebrook 7H9 broth to a total volume of 5 mL. The mixture was incubated with agitation, and 100 μL aliquots were collected every 10 min over a 1-h period. Each aliquot was immediately centrifuged at 13,800 g for 2 min. The titer of non-adsorbed phage in the supernatant was evaluated using the double-layer agar plate method (Kropinski et al., 2009) as described previously. The experiment was carried out in three biological replicates.

The one-step growth curve of Vic9 phage was carried out as previously described (Gan et al., 2014), with modifications. M. smegmatis mc(2)155 in mid-log phase and Vic9 phage were mixed at a MOI of 0.01 in a total volume of 0.9 mL and incubated at 37°C for 30 min to allow phage adsorption. To inactivate unbound phages, 0.1 mL of 100 mM ferrous ammonium sulfate (Sigma-Aldrich, USA) was added to the mixture, following the optimal concentration previously demonstrated to be most effective for phage inactivation (McNerney et al., 1998). After incubation at room temperature for 5 min, the mixture was centrifuged at 13,800 g for 2 min. The pellet was resuspended in 1 mL of Middlebrook 7H9 broth, and the resulting suspension was used to determine the number of infected cells and to construct a one-step growth curve.

The number of infected cells was determined using the double-layer agar plate method (Kropinski et al., 2009) with modifications. Briefly, an aliquot of the suspension, diluted from 10^–1 to 10^–7, was added to soft agar, which additionally contained M. smegmatis mc(2)155 in mid-log phase for better visualization. The number of plaques formed was equal to the number of infected cells.

To construct the one-step growth curve, the resulting suspension (100 μL) was additionally diluted 100-fold in Middlebrook 7H9 broth and incubated with agitation. Aliquots were collected every 30 min for 5 h and centrifuged at 13,800 g for 2 min. The titer of free phage in the supernatant was assessed using the double-layer agar plate method (Kropinski et al., 2009). The experiment was carried out in three biological replicates.

The burst size was determined as the number of phages released from each infected cell, calculated by the ratio of the final number of released phage particles to the initial number of infected bacterial cells.

Phage activity against mycobacterial strains was determined by spot testing on double-layer agar (Sarkis and Hatfull, 1998). For this, 1 mL of the test bacterial culture suspension was washed twice with fresh Middlebrook 7H9 broth to remove Tween-80. The pellet was resuspended in 100 μL of Middlebrook 7H9 broth and added to 5 mL of soft agar and poured onto Middlebrook 7H11 agar. Tenfold serial dilutions of the phage in MP buffer (initial titer of 10⁹ PFU/mL) were spotted in 5 μL drops on the surface of the agar. The plates were incubated for 48 h for fast-growing mycobacteria and 3 weeks for slow-growing ones. The efficiency of plating (EOP) was calculated as the ratio of the phage titer on the test strain to that on the host strain M. smegmatis mc(2)155.

Phage genomic DNA was extracted from the lysate using the standard phenol-chloroform extraction protocol (Green et al., 2012). The extracted DNA (100 ng) was used for library preparation using the KAPA HyperPlus Kit (Roche, Switzerland) according to the manufacturer’s protocol. The library underwent a final cleanup using KAPA HyperPure Beads (Roche, Switzerland), after which the library size distribution and quality were assessed using a high sensitivity DNA chip (Agilent Technologies). The libraries were then quantified using the Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific). The DNA libraries underwent sequencing using the HiSeq 2,500 platform (Illumina, USA), in accordance with the manufacturer’s recommendations. For this purpose, following reagent kits were employed: HiSeq Rapid PE Cluster Kit v2, HiSeq Rapid SBS Kit v2 (200 cycles), and HiSeq Rapid PE FlowCell v2. Additionally, a 2% PhiX spike-in control was included in the process.

Vic9 was isolated from soil samples collected at a livestock yard in the Moscow region, where small ruminants (goats) were kept. The phage plaques on the host strain M. smegmatis mc(2)155 were 1–3 mm in diameter, with clear edges, a round shape, and a turbid center (Figure 1A). Electron microscopy revealed that Vic9 exhibits typical siphophage morphology, characterized by an icosahedral head, 68 ± 2 nm in diameter, and a long non-contractile tail, 265 ± 10 nm in length (Figure 1B).

The optimal MOI assay of the Vic9 indicated that the highest titer, 6.9 × 10⁹ PFU/mL, was achieved at a MOI of 0.01. The adsorption time of the phage to M. smegmatis mc(2)155 was approximately 30 min (Figure 2A). One-step growth curve analysis showed that Vic9 has a latent period of 90 min, followed by a rise period lasting 150 min (Figure 2B), with an estimated burst size of approximately 68 PFU per infected cell.

To determine the host range, serial dilutions of the Vic9 phage were tested on various Mycobacterium species (Figure 3). The phage displayed a narrow host range, effectively lysing only the host strain and M. tuberculosis H37Rv. However, in contrast to M. smegmatis mc(2)155, the phage produced small, irregularly shaped, translucent plaques with indistinct borders on M. tuberculosis H37Rv. No lysis was observed on M. kansasii, M. avium, M. fortuitum, or M. abscessus, regardless of the phage titer.

The complete genome of the Vic9 phage consists of linear double-stranded DNA, 67,543 bp in length, with a G + C content of 69%. A total of 89 open reading frames (ORFs) were identified, covering 63,351 bp (94% of the genome).

BLASTn analysis revealed that Vic9 shares a high degree of similarity with other phages of subcluster B2, showing over 95% query coverage and 98.9% identity. To verify its taxonomic position, a phylogenetic analysis of the phage, along with phages of cluster B (n = 375) from the actinobacteriophage database PhagesDB (Russell and Hatfull, 2017), was conducted. Phylogenetic analysis confirmed its affiliation with subcluster B2, with Vic9 forming a distinct group with the phages Godines (KR997932.1), Ares (JN699004.1), Qyrzula (DQ398048.1), and Laurie (KX443696.1) (Figure 4).

The genomic organization of phage Vic9 is typical of subcluster B2. Functional analysis categorized its proteins into four main groups: (1) Structure and assembly (16 ORFs); (2) Host cell lysis (2 ORFs); (3) Nucleic acid metabolism and other functions (15 ORFs); and (4) Hypothetical proteins (56 ORFs) (Figure 5). The structural genes are located in the left region of the genome and are oriented on the positive strand. This module comprises several key genes, including those encoding the terminase (Vic_00007), RuvC-like resolvase (Vic_00009), portal protein (Vic_00012), MuF-like protein (Vic_00014), major head protein (Vic_00015), major capsid protein (Vic_00016), major tail protein (Vic_00021), head-tail adaptor (Vic_00024), tail assembly chaperone (Vic_00027), tape measure protein (Vic_00029), minor tail proteins (Vic_00030-Vic_00033), head protein (Vic_00034), and virion structural protein (Vic_00042). The tape measure protein, Vic_00029, plays a crucial role in determining tail length and is composed of 1,880 amino acid residues, consistent with the observed morphology of the phage (Belcaid et al., 2011). The cell lysis module, responsible for completing the lytic cycle and releasing phage particles from the host, contains only lysin A (Vic_00058) and holin (Vic_00059).

Preceding the structural module located in the left region of the genome, a group of genes involved in nucleic acid metabolism and other functions were identified, including genes for queuosine biosynthesis from GTP. These genes include queuine tRNA-ribosyltransferase (Vic_00002), Pre-Q0 pathway QueC-like protein (Vic_00003), QueD-like 6-pyruvoyl-tetrahydropterin synthase (Vic_00004), QueE-like queuosine biosynthesis protein (Vic_00005), and GTP cyclohydrolase I (Vic_00006). Additional genes related to nucleic acid metabolism are mainly located in the right region of the genome and include DNA-binding proteins (Vic_00043, Vic_00044), DNA helicase (Vic_00050), DNA polymerase/primase (Vic_00054), DNA polymerase I (Vic_00057), Rnase E (Vic_00065), HNH endonucleases (Vic_00070, Vic_00082), and a putative elongation factor (Vic_00088).

A distinct feature of B2 subcluster members, including Vic9, is the absence of integrase, RNA polymerase, lysin B, the small terminase subunit, and tRNA-coding genes (Hatfull, 2010).

Comparison of the nucleotide sequence of Vic9 with other mycobacteriophages of subcluster B2 from the NCBI database revealed several distinct features (Supplementary Figure S1). Vic9 contains a unique 435 bp sequence located between the genes Vic_00005 (QueE-like queuosine biosynthesis protein) and Vic_00006 (GTP cyclohydrolase I). This sequence lacks open reading frames when annotated with GeneMarkS v4.32. However, analysis using Pharokka 1.7.2 revealed that this region encodes a protein of unknown function. Notably, no homologs of this sequence or the putative protein it encodes were identified in the NCBI database. Additionally, Vic9 possesses the gene Vic_00055, encoding a protein of unknown function, homologous to FDI79_gp56 (96.15%) from phage Godines (KR997932.1) and J3996_gp55 (98.08%) from phage Laurie (KX443696.1), but absent in other cluster members. Additionally, Vic9 is missing the region between Vic_00087 and Vic_00088, which encodes a hypothetical protein in other mycobacteriophages. Notably, this deletion is also present in the phages Laurie (KX443696.1), Arbiter (JN618996.1), Phantasmagoria (ON970587.1), Holeinone (MG812490.1), and Qyrzula (DQ398048.1).

Further genomic analysis revealed that the structural cassette region Vic_0033-Vic_0035, comprising a minor tail protein, a head protein, and a protein of unknown function, shares homology with the corresponding regions in phages Godines (KR997932.1), Ares (JN699004.1), Qyrzula (DQ398048.1), and Laurie (KX443696.1). This homology places Vic9 on a distinct branch within subcluster B2. Additionally, Vic_0033-Vic_0035 showed significant homology with phages from subcluster B3, as indicated by the analysis of 28 out of 44 genomes from PhagesDB, which displayed over 75% similarity. Notably, no homology with other subcluster B2 members was observed (Figure 6).