vB_BcM_Sam46 and vB_BcM_Sam112, members of a new bacteriophage genus with unusual small terminase structure

One of the serious public health concerns is food contaminated with pathogens and their vital activity products such as toxins. Bacillus cereus group of bacteria includes well-known pathogenic species such as B. anthracis, B. cereus sensu stricto (ss), B. cytotoxicus and B. thuringiensis. In this report, we describe the Bacillus phages vB_BcM_Sam46 and vB_BcM_Sam112 infecting species of this group. Electron microscopic analyses indicated that phages Sam46 and Sam112 have the myovirus morphotype. The genomes of Sam46 and Sam112 comprise double-stranded DNA of 45,419 bp and 45,037 bp in length, respectively, and have the same GC-content. The genome identity of Sam46 and Sam112 is 96.0%, indicating that they belong to the same phage species. According to the phylogenetic analysis, these phages form a distinct clade and may be members of a new phage genus, for which we propose the name ‘Samaravirus’. In addition, an interesting feature of the Sam46 and Sam112 phages is the unusual structure of their small terminase subunit containing N-terminal FtsK_gamma domain.

www.nature.com/scientificreports/ syndrome caused by the toxin cereulide 19 . B. cytotoxicus is the less studied species than those mentioned above inasmuch as it has been fairly recently described for the first time 20 . B. cytotoxicus is responsible for severe diarrheal disease due to production of its cytK2 variant of cytotoxin K, which is able to cause necrotic enteritis 21 . Contamination of foodstuffs with spores and/or vegetative cells of both B. cereus and B. cytotoxicus poses a serious threat to public health and can cause extensive harm to food industry and agriculture. B. thuringiensis is referred to as an entomopathogenic species able to produce crystal insecticidal toxins ( δ-endotoxins) 22 . This is why biopesticidal strains of B. thuringiensis have become widespread as microbiological biopesticides being the most environmentally friendly and safe for human health among insecticidal products available 23,24 . Nevertheless, the actual contribution of B. thuringiensis to the cases of poisoning is still highly contentious 25,26 . Taking into account the major challenge of the emergence and spread of multi-drug resistant bacteria including pathogens of the B. cereus group 27,28 , it is apparent that phage therapy has the potential to become either an alternative or a supplement to the classical antibiotic treatments. However, the creation of effective phage-based preparations requires developing and maintaining the collections of carefully selected and well-studied bacteriophages acting on target pathogen species and strains.
In this study, we describe the newly isolated and characterized Bacillus phages vB_BcM_Sam46 and vB_ BcM_Sam112 (abbreviated as Sam46 and Sam112, respectively), which effectively infect members of the B. cereus group. Phages Sam46 and Sam112 have morphological characteristics typical of myoviruses. The genomes of Sam46 and Sam112 comprise double-stranded DNA, 45,419 bp and 45,037 bp in length, respectively, and the same GC-content. The genome identity of Sam46 and Sam112 is 96.0% indicating that they belong to the same phage species. According to the phylogenetic analysis, these phages form a distinct clade and may be members of a new phage genus. We propose to create a new bacteriophage genus called 'Samaravirus' to formally classify these phages.

Materials and methods
Bacterial strains and growth conditions. The bacterial strains used in this study were obtained from the All-Russian Collection of Microorganisms (VKM). Lysogeny broth (LB) and LB agar (1.5% w/v and 0.75% w/v) with 10 mM CaCl 2 and 10 mM MgCl 2 were used for bacterial and phage cultivation. All cultures were grown at 37 • C.
Phage isolation and propagation. Phages Sam46 and Sam112 were isolated from soil samples collected in Samara, Russian Federation, and propagated on the sensitive strain B. cereus VKM B-370. The sensitive strain B. cereus VKM B-370 was grown in 10 ml of LB broth with 10 mM CaCl 2 and 10 mM MgCl 2 to the optical density of 0.6 at 590 nm. One gram of the soil sample was added to the cell culture and the mixture was incubated for 2 h at 37 • C until optical density decreased. The obtained suspension was centrifuged for 10 min at 12,000g. After that, the lysate was mixed with 100 µ l of B. cereus VKM B-370 culture (OD590 of 0.35) and 1.5% agar to the final agar concentration of 0.5%. After gentle short-term vortexing, the mixture was poured into Petri dishes with previously prepared LB agar (1.5%) and incubated overnight at 37 • C. Further, the separate plaques were transferred into 2 ml of SM+ buffer [125 mM Tris-HCl, pH 8.0; 100 mM NaCl; 2.5 mM MgSO 4 ; 0.01% gelatin; 10 mM CaCl 2 ] and incubated overnight at 4 • C with shaking for phage extraction. The extracts of single plaques were centrifuged for 10 min at 12,000g, and 50 µ l of the supernatants were transferred into a 48-well plate containing 500 µ l of LB broth (with additional 10 mM CaCl 2 , 10 mM MgCl 2 ) with 5 µ l of B. cereus VKM B-370 culture (OD590 0.6) and incubated for 16 h at 37 • C until optical density decreased. The cultures with the lowest optical densities were transferred into 1.5 ml Eppendorf tubes with 50 µ l of chloroform. The cell debris was removed by centrifugation for 10 min at 12,000g. The obtained lysates were titrated by serial dilutions. In order to exclude the presence of other phages with morphologically identical plaques, the extraction-titration cycles were repeated five times. Phage propagation and PEG 8,000 (polyethylene glycol) precipitation were performed as described previously 29 . The obtained high-titer phage sample was filtered through a 0.22 µ m sterile filter and stored at 4 • C. Three ml of the high-titer preparation was subsequently used to prepare the final purified phage suspension by CsCl density gradient centrifugation (with preformed gradient of CsCl: 1.3 g/ml, 1.4 g/ml, 1.5 g/ ml, 1.6 g/ml and 1.7 g/ml). The purified suspension was ultimately used for transmission electron microscopy.
Host range determination. A host range test was performed using 32 strains of the B. cereus group as described previously 29 with differences in incubation conditions (for 24 h at 37 • C).
Transmission electron microscopy. Phage suspension applied onto 400 mesh carbon-formvar coated copper grids was negatively stained with 1% uranyl acetate and subsequently analyzed using a JEM-100C (JEOL, Japan) transmission electron microscope at 80 kV accelerating voltage. Images were taken on Kodak film SO-163 (Kodak, Cat. # 74144, Hatfield, PA, USA). Phage particle dimensions were measured using ImageJ version 1.53e in relation to the scale bar generated from the microscope.
Genome sequencing, assembly and sequence analysis. Phage DNA was extracted using the standard phenol-chloroform extraction protocol described by Sambrook et al. 30 . Purified phage DNA was sequenced using Illumina with TruSeq library preparation technology. The de novo genome assembly was accomplished by SPAdes v.3.11.1 software 31 . Open reading frames (ORFs) were identified by RASTtk v.2.0 32 . The putative functions were predicted using BLAST (NCBI) 33  Headful DNA analysis. The bacteriophage genome termini were identified based on the read occurrence frequency following the High Occurrence Read Termini theory 43,44 . Mapping the reads to the genome was carried out with the Bowtie2 software tool v.2.3.4.3 45,46 . The in silico results were experimentally confirmed using a standard restriction analysis 47 . The pac-site was determined by sequencing terminal DNA fragments obtained by the method of rapid amplification of genomic ends (RAGE) 48 , which is based on the principles of the 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE). For this purpose, the specific fragments suspected to contain the phage genome termini were extracted from electrophoresis gel after restriction analysis and used for a typical DNA tailing reaction by terminal transferase (New England Biolab, Cat. # M0315L). Further, two PCRs were carried out consecutively using the TaqSE  Identification of genetic differences between phages with turbid and clear plaque morphotypes. In order to identify genetic differences between the Sam46 types forming turbid and clear plaques (Sam46-T and Sam46-C, respectively), separate plaques with different morphology were used for T-type and C-type phage extraction followed by obtaining high-titer phage samples, phage DNA extraction and whole genome sequencing (WGS) as mentioned above.
To obtain mutant phages Sam46-C and Sam112-C from Sam46-T and Sam112-T, respectively, T-type phages were propagated in 30 ml of LB broth (10 mM CaCl 2 , 10 mM MgCl 2 ) inoculated with 300 µ l of B. cereus VKM B-370 culture (OD590 0.6). Cultivation was continued at 37 • C until optical density decreased. The cell debris was removed by centrifugation at 12,000g for 10 min at 4 • C. Phage particles were precipitated from the obtained lysates with PEG 8,000 as described previously 29 . The propagation-precipitation cycles were repeated three times. Then, 10 µ l of the final concentrated T-type phage suspensions diluted to 10 4 plaque-forming units (PFU)/ml were mixed with 100 µ l of B. cereus VKM B-370 culture (OD590 0.6) and 5 ml of LB top agar (0.75%) and overlaid on LB agar plates (1.5% w/v agar) in Petri dishes. Incubation was performed at 37 • C overnight for the formation of phage plaques and detection of turbid-to-clear plague mutations. Thus, 40 plates were analyzed for each T-type phage (Sam46-T and Sam112-T). For each phage, three clear plaques were selected for further manipulations. The selected mutated plaques were separately transferred into 50 µ l of Milli-Q water and incubated for 15 min at 95 • C. Two microliters of each mixture were used as PCR templates. PCRs were carried out consecutively using the TaqSE DNA polymerase (SibEnzyme, Cat. # E314) and the pair of oligonucleotides: Sam46-112_CDS_24_Fw 5 ′ -TCT ATT TTC AAA GCA AGC GG-3 ′ and Sam46-112_CDS_26_Rv 5 ′ -GCT AAT TTC TTA ACC GGT TC-3 ′ . PCR products were extracted from electrophoresis gel and used for Sanger sequencing with the primer Sam46-112_CDS_24_Fw 5 ′ -TCT ATT TTC AAA GCA AGC GG-3 ′ .
The phage mutants with turbid plaques from Sam46-C and Sam112-C were obtained similarly.
Prediction of protein secondary structures. Secondary structure prediction of the gp25 gene products of Sam46 and Sam112 phages was performed with JPRED4 incorporating the Jnet algorithm v2.3.1 49 . The oligomeric state probabilities of a coiled-coil sequence were predicted by LOGICOIL algorithm 50 .
Thermal and pH stability of the Sam46-T and Sam46-C phages. In  www.nature.com/scientificreports/ Killing assay. To assess the killing activity of the Sam46-C and Sam46-T phages, the B. cereus VKM B-370 strain was separately infected with the Sam46-C and Sam46-T phages at different multiplicity of infection (MOI) values. Briefly, 50 µ l of phage suspensions ( 2 × 10 9 , 2 × 10 8 and 2 × 10 7 PFU/ml) were mixed separately in a 48-well microplate with 450 µ l of mid-log B. cereus VKM B-370 culture at a concentration of 2 × 10 7 colonyforming units (CFU)/ml to provide MOI values of 10, 1, and 0.1, respectively. The microplate was incubated with shaking for 7 h at 30 • C in a FilterMax F5 microplate reader (Molecular Devices), with OD595 being measured every 10 min. Non-infected B. cereus VKM B-370 culture was used as a control sample. The experiment was performed with three replicates. The results were reported as the mean of three observations ± standard deviation. The obtained growth curves were visualized in SigmaPlot v.12.5.
Adsorption assay. To determine the time required for the Sam46-T and Sam46-C phages to attach to B.
cereus VKM B-370 cells, an adsorption assay was performed according to the protocol developed by Kropinski 51 . Briefly, 0.95 ml of LB broth with three drops of chloroform were added to Eppendorf tubes and placed on ice to chill for 10 min. Mid-log phase B. cereus VKM B-370 culture (OD590 0.4) grown in LB broth with the addition of 10 mM CaCl 2 and 10 mM MgCl 2 was diluted to OD590 of 0.2. Nine milliliters of the culture were transferred to a 100-ml laboratory flask and the flask was placed into a shaking water bath at 37 • C, 60 rpm. Nine milliliters of LB broth with 10 mM CaCl 2 and 10 mM MgCl 2 were used as a control sample. Immediately, 1 ml of the Sam46-T or Sam46-C phage suspension at a concentration of 2 × 10 5 PFU/ml (preheated for 5 min at 37 • C) was added to both tested and control flasks. Every 5 min, 50-μl aliquots were collected from both tested and control flasks, transferred into the prepared Eppendorf tubes containing LB and chloroform, and mixed vigorously in a vortex mixer. The mixtures were assayed for unadsorbed phages by double-layer plate titration, and the resulting phage titers were compared to those obtained for the control samples (without host cells). This experiment was performed three times for each phage (Sam46-T and Sam46-C). The results were presented as percentages of the initial phage number and visualized in SigmaPlot v.12.5 with error bars representing standard deviation for three replicates. The adsorption rate was calculated using the equation described by Kropinski 51 .
One-step growth curve. In order to determine the latent period and the average burst size of the Sam46-T and Sam46-C phages, the one-step growth experiment was performed as described by Hyman and Abedon 52 . Briefly, 1ml of phage suspension (preheated for 5 min at 37 • C) at a concentration of 2 × 10 7 was mixed with 9 ml of the B. cereus VKM B-370 culture grown to the mid-log phase (OD590 0.4) and diluted to OD590 of 0.2 to provide MOI of 0.1. After the incubation at 37 • C for 10 min for phage adsorption, a 1-ml aliquot was collected and centrifuged at 3,500g for 10 min at 4 • C to precipitate the cells. The supernatant was removed and the pellet was resuspended in 1 ml of fresh LB broth, centrifuged and resuspended in 1 ml of LB again. 0.1 ml of the mixture (bacteria with adsorbed phages) was transferred to 9.9 ml of LB growth medium in a 100-ml flask, mixed thoroughly and placed in a shaking water bath (60 rpm) at 37 • C. Aliquots of 150 µ l of the mixture were collected at 5-min intervals for 1 h. The phages were enumerated by double-layer plate titration at each time point, including time point 0. The experiment was performed in triplicate for each phage (Sam46-T and Sam46-C). The PFU/ml values were calculated and plotted against time. The latent period and phage burst size were calculated from the plotted curve visualized in SigmaPlot v.12.5 with error bars representing standard deviation for three replicates.
Phage immunity test. www.nature.com/scientificreports/ of plaques were obtained after five cycles of the extraction-titration procedure. Thus, four phages were ultimately obtained: Sam46-C, Sam46-T, Sam112-C and Sam112-T. The letters C and T indicate the morphotypes of plaques formed by the phages: clear and turbid morphotype, respectively. A host range test was carried out for 32 strains of the B. cereus group. Both original and purified (producing uniform plaques) suspensions of Sam46 and Sam112 were capable of forming plaques on the lawns of 17 and 16 (out of 32) strains ( ∼ 50%) , respectively ( Table 1). The TEM analysis revealed that both Sam46 and Sam112 (for each phage, n = 10 phage particles) possess an icosahedral non-elongated head of approximately 57.62 ± 1.2 nm in diameter attached to a characteristic long contractile nonflexible tail of approximately 160.0 ± 5.5 nm in length (including the baseplate structure) and 16.9 ± 1.3 nm in width, ending with a baseplate structure with six tail fibers. These phages have the characteristic morphological features of the myovirus morphotype (Fig. 1).
General genome organization of Sam46 and Sam112. The Sam46 genomic sequence contains 45,419 bp with the GC-content of 41.6% and 77 predicted ORFs. The Sam112 genome is slightly shorter, with 45,037 bp, 75 predicted ORFs and the same GC-content. According to the BLASTn (NCBI), these phages have the whole genome identity value of 96.0% ( Supplementary Information, Table S3), indicating that they belong to the same phage species in accordance with the official ICTV classification, as the currently used species demarcation criterion is the genome nucleotide identity of 95% 53,54 . The circular genome map of Sam46 is shown in Fig. 2, with the first base of small terminase subunit gene selected as the starting point of the genome.
The putative functions were assigned to 44 (57.1%) and 44 (58.7%) ORFs of Sam46 and Sam112, respectively, using BLASTp (NCBI) 33 and HHpred 34 . More detailed information about the predicted ORFs is provided in Tables S1 and S2 ( Supplementary Information, Table S1, Table S2).     (Fig. 3a) has revealed that the DNA binding sites of FtsK_gamma domains of both phages are highly conserved among bacteria. The phylogenetic analysis of FtsK_gamma domains indicated that those from B. cereus and B. subtilis are most closely related to the FtsK_gamma domains of Sam46 and Sam112 (Fig. 3b). Therefore, the FtsK_gamma domains of the small terminase subunits of Sam46 and Sam112 were probably acquired from bacterial hosts during co-evolution.
Atypical domain structure of the small terminase subunits of phages Sam46 and Sam112 indicates that the Sam46 and Sam112 genomes might contain KOPS/SRS-like binding sites for the small terminase.
Replication and recombination genes. The module of replication-and recombination-related genes of Sam46 resembles that of the virulent Bacillus bacteriophage SPP1 (Fig. 2) and belongs to the "initiator-helicase-helicase loader" type according to the classification proposed by Weigel and Seitz 62 . It includes the adjacently encoded DnaD domain-containing putative replication initiator (Sam46_gp61; Sam112_gp58), lambda P-related helicase loader (Sam46_gp62; Sam112_gp59) and DnaB-type replicative helicase (Sam46_gp63; Sam112_gp60). Sam46_gp47 (Sam112_gp60) and Sam46_gp49 (Sam112_gp47) encode products with apparent homology to YqaJ domain-containing exonuclease and RecT-like protein, respectively, which thereby seem to be parts of the two-component recombination system functionally similar to SPP1 Gp34.1 63 and Gp35 64 . Sam46_gp46 (Sam112_gp48) is an E. coli-type single stranded DNA binding (SSB) protein known to be frequently associated with such a recombination system 62,65 . Sam46_gp53 (Sam112_gp50) is the processivity factor (DNA sliding clamp) of PolIIIβ-type, which is most abundant among bacteriophages 62 .

Comparative genomics.
To assess the phylogenetic relationship of the Sam46 and Sam112 to known phages, ViPTree server version 1.9 was used to generate a proteomic tree based on the genome-wide sequence similarities computed by tBLASTx (Fig. 4). As can be seen in Figure 4, Sam46 and Sam112 form a separate branch significantly distant from the closest relatives. The most closely related genomes belong to bacteriophages SPP1 and GBK2, which are both siphoviruses.
In addition, the number of shared proteins was computed using the GET_HOMOLOGUES software (Supplementary Information, Table S4), and a pangenome matrix was produced showing the presence/absence of representatives of individual gene/protein clusters in each genome. Based on the matrix, a Maximum-likelihood (ML) tree was drawn (Fig. 5), where the intergenomic distances are computed from the number of shared proteins. According to Table S3 and Fig. 5, the myovirus D6E shares slightly more proteins (20) than SPP1 (16) with Sam46. Thus, the VipTree and GET_PHYLOMARKERS phylogenetic analyses failed to give consistent results, which is not in any way surprising: both SPP1 and D6E share less than 23% of their proteins with Sam46, therefore, for the question which one of them is the closest relative to make sense, both are far too distant.
The linear whole genome comparison diagram was also visualized with the ViPTree server version 1.9 (Fig. 6), showing tBLASTx pairwise similarities between the most closely related genomes. As illustrated on the diagram, both the SPP1 and D6E genomes contain regions of local similarity to the Sam46 and Sam112 genomes, although   (Fig. 7a), which is typical of phages with circularly permuted terminal repeats using the headful mechanism of DNA packaging 37 . The DNA packaging mode of Sam46 and Sam112 was experimentally confirmed by restriction analysis of the genomic DNA using restriction endonucleases HindIII, XbaI and HindIII with XbaI simultaneously. For both Sam46 and Sam112, on each track of the electrophoregrams we could find all of the fragments predicted in silico from artificially circularized genomic sequences and also an additional unexpected fragment appearing in submolar concentrations relative to others (Fig. 7b) 66 . This type of restriction pattern is known to be characteristic of phages with the headful mode of DNA packaging 66 . The additional submolar fragments are so-called "pac-fragments", which are produced during the first packaging event at each genomic concatemer and, unlike true restriction fragments, are cut on one side by the phage terminase. The observed lengths of the pac-fragments allowed us to predict the approximate location of the pac-site, which is expected to be roughly 4.5 Kbp upstream of the TerS gene in both Sam46 and Sam112 genomes. The Sam46 pac-fragments were extracted from the electrophoresis gel and used for determination of the pacsite location using the RAGE method followed by Sanger sequencing of the products obtained in the second PCR. For the HindIII-and XbaI-generated pac-fragments, the terminase-generated cut was found to be approximately at position 40,843 in the intergenic region upstream of Sam46_gp66, as is shown on the sequencing chromatograms (Fig. 7c). Thus, both Sam 46 and Sam112 apparently use the headful mechanism of DNA packaging with accurate site-specific initiation.
Identification of genetic differences between the phages with turbid and clear plaque morphotypes. DNA was extracted from the Sam46-T and Sam46-C phages as described in "Materials and methods".
The whole genome sequencing of the Sam46-T and Sam46-C genomes showed differences in the genetic content of only one gene. The Sam46-T genome is completely identical to the originally sequenced Sam46 genome, while the Sam46-C genome contains mutations in the gp25 gene (gene locus_tag: Sam46_gp25; protein ID: QIQ61226.1) encoding XkdW-like protein with unknown function (Fig. 8a).
The series of experiments on obtaining mutant phages with clear plaque morphotype Sam46 and Sam112 from Sam46-T and Sam112-T, respectively, followed by Sanger sequencing of the gp25 gene, confirmed the appearance of point mutations within this gene in C-type phages (Fig. 8a). The frequency of turbid-to-clear plaque mutations in both Sam46 and Sam112 was 1:3000.
All mutations identified in both WGS and the experiment on obtaining mutant C-type phages were found to be located strictly in the C-terminal region of the XkdW-like protein (Fig. 8a,b). Secondary structure prediction www.nature.com/scientificreports/ by JPRED4 49 and coiled-coil region prediction by LOGICOIL 50 have shown that the mutation region is an α -helix containing HPPHPPP patterns, referred to as heptad repeats (abcdfg) 67 , where hydrophobic residues (H) generally occupy the "a" and "d" positions, and polar residues (P) occur on other positions (Fig. 8b,c). Such a structure of α-helix is characteristic of the coiled-coil motif in proteins. According the LOGICOIL prediction 50 , the most probable state of coiled coils of XkdW-like proteins of both Sam46 and Sam112 is a parallel dimer. The experiments on obtaining mutant phages with the turbid plaque morphotype from Sam46-C and Sam112-C showed no results. Perhaps, the probability of the clear-to-turbid transition is extremely low.
Thermal and pH stability of the Sam46-T and Sam46-C phages. Taking into account the fact that the Sam46 and Sam112 phages are the same species according to the genome-wide analysis (the whole genome identity of 96.0%), this and the subsequent experiments were performed only with Sam46 as a representative of this species. In an attempt to explain the origin of two types of plaques, we performed the experiment for both Sam46-T and Sam46-C phages.
Both Sam46-T and Sam46-C phages were stable at temperatures of 30, 40 and 50 • C, as phage titers were similar to those of the control samples incubated at 4 • C (Fig. 9a,b). At 60 • C or higher, the phage titer dropped sharply, no viral particles were detected after 1 h of incubation, and phage activity was completely lost (Fig. 9a,b). In addition, the results of pH stability tests showed that both Sam46-T and Sam46-C phages were stable at pH values from 5 to 10 (Fig. 9c,d). However, the Sam46-T and Sam46-C phages are not able to survive under highly acidic conditions, as no phages survived incubation in solutions at pH 2.2, 3 and 4.  Supplementary Information, Fig. S1) may have different explanations, the simplest one being the formation of lysogens. However, in the 'Phage immunity test' section below we show that it does not appear to be the case. www.nature.com/scientificreports/ Phage immunity test. As has been mentioned above, the difference between the Sam46-C and Sam46-T plaque morphotypes ( Supplementary Information, Fig. S1), as well as the difference between the rates of B. cereus VKM B-370 lysis (Fig. 10) upon Sam46-T and Sam46-C infection, may be related to the formation of lysogens. Temperate phages, in the prophage stage, are known to make the host bacterium resistant to viruses identical or closely related to the prophages. This phenomenon is a special case of what is known as 'superinfection exclusion' 68,69 and is widely used to detect the presence of prophages.
To assess the immunity of B. cereus VKM B-370 cells collected from the centre of five turbid plaques (putative lysogens) against Sam46-T and Sam46-C infection, the cells were infected with Sam46-T or Sam46-C, as described in "Materials and methods". The optical density (OD595) of the cultures was measured upon the infection, and the measurement was performed three times for both the original B. cereus VKM B-370 cells and the putative phage-immune cells. As is shown in Fig. 11, the optical density of the putative phage-immune B. cereus VKM B-370 cultures decreased similarly to that of the original B. cereus VKM B-370 strain.
Thus, the appearance of Sam46 and Sam112 plaques with different morphotypes and the difference in the rate of B. cereus VKM B-370 lysis upon T-type and C-type phage infections are not associated with the formation of lysogens.
In view of the above and considering the fact that no genes typical of temperate phages have been found in the Sam46 and Sam112 genomes (see "General genome organization of Sam46 and Sam112"), it can be concluded that the phages are virulent.
Adsorption assay. An adsorption assay was performed to identify the rate at which the Sam46-T and Sam46-C phages are adsorbed to the cell surface of B. cereus VKM B-370. As shown in Fig. 12, a, the adsorption rates of Sam46-T and Sam46-C are highly similar: about 50% and 80% of the phages attach to the host cells within 10 and 20 min, respectively. The adsorption rates of Sam46-T and Sam46-C are 9.68 ± 0.36 × 10 −10 ml/ min and 9.20 ± 0.20 × 10 −10 ml/min, respectively.
One-step growth curve. The growth kinetics of Sam46-T and Sam46-C was determined by the one-step growth curve method. The latent period of both Sam46-T and Sam46-C is about 20 min and the duration of one lytic cycle is 15-20 min. The burst size was found to be 450.5 ± 70.5 PFU and 565.6 ± 64.6 PFU per infected cell for Sam46-T and Sam46-C, respectively (Fig. 12b).    53 . Both Sam46 and Sam112 were able to form two types of plaques: clear (C-type) and turbid (T-type) on the lawn of the host strain B. cereus VKM B-370. The purified C-type and T-type phages were able to stably produce uniform plaques in the following generations. As has been shown, the appearance of Sam46 and Sam112 plaques with different morphotypes and the difference in the rate of B. cereus VKM B-370 lysis upon T-type and C-type phage infections are not associated with the formation of lysogens (Fig. 11). The genome-wide analysis of C-type and T-type Sam46 phages suggests that the phenotypic difference of plaques is due to mutations in the gp25 gene encoding the XkdW-like protein with unknown function. The structural analysis of the XkdW-like protein showed that the detected mutations were located in the coiled-coil sequence (Fig. 8). The coiled-coil motifs are found in numerous protein structures including phage structural proteins such as fibritin (gpwac) of bacteriophage T4 70,71 and the distal subunit of the long tail fiber (gp37) 72 of bacteriophage T4. The coiledcoil regions of such phage proteins proved to be crucial for the folding of the proteins, as well as for promoting conformational changes and contributing to protein-protein interactions [70][71][72] . Based on our experimental data and taking into account the gp25 gene neighboring tail-related genes (gp24, gp26), as well as considering the structural features of the XkdW-like protein it encodes, we suggest that the gp25 product is apparently involved in phage tail assembly. With this in mind, the XkdW-like protein may be related to phage adsorption or may affect characteristics of the virion assembly such as the rate of assembly and the number of active phage particles. Different adsorption rates of T-type and C-type phages would explain the phenomenon of morphologically different plaques. However, the adsorption assay did not lend credence to this hypothesis. As shown in Fig. 12 a, about 80% of the Sam46-T and Sam46-C particles are adsorbed to the host cells within 20 min, with the rates of adsorption being 9.68 ± 0.36 × 10 −10 ml/min and 9.20 ± 0.20 × 10 −10 ml/min, respectively. According to the results of the one-step growth curve experiment, the burst sizes of the Sam46-T and Sam46-C phages are slightly different: approximately 450.5 ± 70.5 PFU and 565.6 ± 64.6 PFU per infected cell, respectively. Thus, more detailed studies are needed for clear understanding of the XkdW-like protein function and its role in the Sam46 and Sam112 lifecycles.
The thermal and pH stability of Sam46-T and Sam46-C were determined. Both Sam46-T and Sam46-C phages are stable at the temperatures ranging from 4 to 50 • C, and a further increase in temperature (60 • C and higher) results in the complete inactivation of the phages (Fig. 9a,b). Almost 100% of both Sam46-T and Sam46-C phages survived at pH values ranging from 5 to 10, but neither phage survived at pH 2.2, 3 and 4 (Fig. 9c,d).
The predicted headful DNA packaging mechanism of Sam46 and Sam112 was experimentally confirmed by restriction analysis (Fig. 7b). We also determined the pac-site location using the method RAGE 48 followed by Sanger sequencing (Fig. 7c).
An interesting feature of the Sam46 and Sam112 phages is the unusual structure of their small terminase subunit initiating DNA packaging. The small terminase subunit of these phages possesses a two-domain structure with the typical C-terminal Terminase_2 domain (residues 107-230) and N-terminal FtsK_gamma domain (residues ; the latter has not been previously described as part of the small terminase subunit. Some phage genomes have been shown to contain genes encoding FtsK_gamma-like proteins, the role of which is still unclear. FtsK_gamma domain is a well-studied component of bacterial motor proteins such as the FtsK protein of E. Figure 12. (a) Adsorption assay and (b) Growth parameters of the Sam46-T and Sam46-C phages. The graphs was created with SigmaPlot v.12.5 (http:// www. sigma plot. co. uk/ produ cts/ sigma plot/ produ pdates/ prod-updat es18. php). Error bars represent standard deviation for three replicates.