Morphology characterization and biological features of phage vB_Bsu_hmny2
We observed clear plaque halos on agar plates when replicating phage vB_Bsu_hmny2 using B. subtilis WB800 as the host, with plaques measuring approximately 1–3 mm in diameter. The morphology of phage vB_Bsu_hmny2 was illustrated in Fig. 1A, and its structure as seen under TEM was depicted in Fig. 1B. The phage particles possessed a head with a diameter of 40 ± 10 nm and a short tail measuring 20 ± 6 nm. All morphological properties support the assignment of vB_Bsu_hmny2 to the Beecentumtrevirus, which belongs to the Picovirinae subfamily (ICTV 2023). The phages were propagated and purified at a MOI of 0.1, achieving a maximal viral titer of 4.9×1010 PFU/mL.
The optimal MOI for vB_Bsu_hmny2 was identified as 10− 6 through repeated measurements, as illustrated in Fig. 1C. At this MOI, the phage achieved its highest titer. The life cycle of the phage was characterized using a one-step growth curve. Results indicated a latent period of 30 to 60 min, followed by a lysis period of approximately 5 h, during which the phage count surged and then stabilized (Fig. 1D). The burst size was calculated to be 199.5 PFU per cell.
The phage titer remained relatively stable across pH levels from 3 to 12. However, significant titer reduction was observed under acidic conditions, with complete inactivation occurring at pH 2. Similarly, under alkaline conditions, the titer dropped significantly as pH rose from 12 to 13, with total inactivation at pH 13 (Fig. 1E). Heat stability tests revealed no notable decrease in titer between 4°C and 80°C, but the phage was entirely inactivated at 90°C (Fig. 1F).
The efficacy of vB_Bsu_hmny2 against a culture of B. subtilis WB800 was tested. In the phage lysis experiment, vB_Bsu_hmny2 demonstrated effective bacteriolytic activity at MOIs of 1, 0.1, and 0.01 compared to the control group without adding phages. The rate of bacterial lysis increased with the MOI. Specifically, at an MOI of 1, the OD600nm of the host bacterial cells decreased significantly after approximately 80 min, with bacterial levels stabilizing around 3.5 h post-infection. At MOIs of 0.1 and 0.01, the turbidity of B. subtilis decreased to and stabilized at its lowest level after approximately 4.5 h and 6 h, respectively.
Genome sequence analysis
The whole genome length of phage vB_Bsu_hmny2 was 18,762 bp with 37% G + C pair content. Out of the 25 predicted ORFs, 17 ORFs are associated with genes encoding proteins with known functions and 8 ORFs encode hypothetical proteins with unknown functions (Fig. 3).
The annotated functional proteins were divided into several groups depending on their function. The group of DNA-replication-, recombination-, repair-, and packaging-associated genes included five ORFs, encoding (ORF 1, ORF 17, ORF 19, ORF 20, and ORF 21). Eight ORFs were predicted to encode structural proteins (ORF 9 and ORF 16). Two genes encoding proteins associated with transcription, translation, and nucleotide metabolism were predicted: (ORF 5 and ORF 6). Another two proteins associated with lysis, including: endolysin (ORF 7) and holin (ORF 8). The two enzymes are capable of disrupting bacterial peptidoglycan from within, leading to bacterial lysis and the release of new phages. The BLAST search results for these proteins are presented in Table S1. No genes associated with virulence, antimicrobial resistance, or toxins, which would make them unsatisfactory for therapeutic purposes, were identified. No tRNA genes were identifie in the genome of vB_Bsu_hmny2, which suggests that the translation mechanism of this new phage is host-dependent [23, 24].
Phylogenetic analysis
BLASTn analysis revealed that vB_Bsu_hmny2 exhibits high sequence similarity with three previously reported Bacillus phages from the Beecentumtrevirus genus: Nf [25] (coverage: 99%; identity: 96.33%), B103 [26] (coverage: 98%; identity: 91.47%), and vB_BsuP_Goe1 [27] (coverage: 96%; identity: 96.72%). The genomes of these phages range in size from 18,300 bp to 18,800 bp. Taxonomic analysis was performed with ViPTree server. This programme uses the proteomic tree based on genome-wide comparison with the reference genomes including prokaryotic dsDNA viruses in the database. Circular and rectangular trees were generated (Fig. 4). The position of vB_Bsu_hmny2 in the phylogenic tree also indicated that vB_Bsu_hmny2 is a member of Beecentumtrevirus.
Bacillus phage phi29 is a widely studied phage, noted for its extensive applications in molecular biology and genetic engineering [28, 29]. Prior to the dissolution of the Podoviridae family, vB BsuP-Goe1, Nf, and B103 were classified under the genus Phi29likevirus. Comparative analysis with the closely related phage vB_Bsu_hmny2 revealed that this phage harbors a conserved gene set essential for replication. However, variations in the gene sequences and genomic structural compositions are more prevalent at the termini of the genome (Fig. 5).
Previous literature has reported that the early protein 17 (ORF 1) represents a region with high sequence variability [27]. Amino acid sequence alignment indicates that the early protein 17 from phage vB_Bsu_hmny2 shows only 40.74% similarity to the early protein 17 of vB BsuP-Goe1. In contrast, the sequence similarity with early proteins from phages Nf and B103 exceeds 90%.