Identification and characterization of two Bacillus anthracis bacteriophages.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

Characterization of the novel phage vB_BceP_LY3 and its potential role in controlling Bacillus cereus in milk and rice.

Bacillus cereus is a foodborne pathogen that induces vomiting and diarrhea in affected individuals. It exhibits resistance to traditional sterilization methods and has a high contamination rate in dairy products and rice. Therefore, the development of a new food safety controlling strategy is necessary. In this research, we isolated and identified a novel phage named vB_BceP_LY3, which belongs to a new genus of the subfamily Northropvirinae. This phage demonstrates a short latency period and remains stable over a wide range of temperatures (4-60 °C) and pH levels (4-11). The 28,124 bp genome of LY3 does not contain any antibiotic-resistance genes or virulence factors. With regards to its antibacterial properties, LY3 not only effectively inhibits the growth of B. cereus in TSB (tryptic soy broth), but also demonstrates significant inhibitory effects in various food matrices. Specifically, LY3 treatment at 4 °C with a high MOI (MOI = 10,000) can maintain B. cereus levels below the detection limit for up to 24 h in milk. LY3 represents a safe and promising biocontrol agent against B. cereus, possessing long-term antibacterial capabilities and stability.

Phages overcome bacterial immunity via diverse anti-defence proteins.

It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.

Isolation and Characterisation of the Bundooravirus Genus and Phylogenetic Investigation of the Salasmaviridae Bacteriophages.

Bacillus is a highly diverse genus containing over 200 species that can be problematic in both industrial and medical settings. This is mainly attributed to Bacillus sp. being intrinsically resistant to an array of antimicrobial compounds, hence alternative treatment options are needed. In this study, two bacteriophages, PumA1 and PumA2 were isolated and characterized. Genome nucleotide analysis identified the two phages as novel at the DNA sequence level but contained proteins similar to phi29 and other related phages. Whole genome phylogenetic investigation of 34 phi29-like phages resulted in the formation of seven clusters that aligned with recent ICTV classifications. PumA1 and PumA2 share high genetic mosaicism and form a genus with another phage named WhyPhy, more recently isolated from the United States of America. The three phages within this cluster are the only candidates to infect B. pumilus. Sequence analysis of B. pumilus phage resistant mutants revealed that PumA1 and PumA2 require polymerized and peptidoglycan bound wall teichoic acid (WTA) for their infection. Bacteriophage classification is continuously evolving with the increasing phages' sequences in public databases. Understanding phage evolution by utilizing a combination of phylogenetic approaches provides invaluable information as phages become legitimate alternatives in both human health and industrial processes.

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.

Bacillus-infecting bacteriophage Izhevsk harbors thermostable endolysin with broad range specificity.

Several bacterial species belonging to the Bacillus cereus group are known to be causative agents of food poisoning and severe human diseases. Bacteriophages and their lytic enzymes called endolysins have been widely shown to provide for a supplemental or primary means of treating bacterial infections. In this work we present a new broad-host-range phage Izhevsk, which infects the members of the Bacillus cereus group. Transmission electron microscopy, genome sequencing and comparative analyses revealed that Izhevsk is a temperate phage with Siphoviridae morphology and belongs to the same genus as the previously described but taxonomically unclassified bacteriophages Tsamsa and Diildio. The Ply57 endolysin of Izhevsk phage has broad-spectrum activity against B. cereus sensu lato. The thermolability of Ply57 is higher than that of the PlyG of Wß phage. This work contributes to our current understanding of phage biodiversity and may be useful for further development of efficient antimicrobials aimed at diagnosing and treating infectious diseases and food contaminations caused by the Bacillus cereus group of bacteria.

Complete genome sequence of a novel Bacillus phage, P59, that infects Bacillus oceanisediminis.

P59, a virulent phage of Bacillus oceanisediminis, was isolated from the sediment of Weiming Lake at Peking University (Beijing, China). P59 showed the typical morphology of myovirids. The complete genome sequence of P59 is 159,363 bp in length with a G+C content of 42.34%. The genome sequence has very low similarity to the other phage genome sequences in the GenBank database, suggesting that P59 is a new phage. A total of 261 open reading frames and 15 tRNA genes were predicted. Based on its morphological and genetic traits, we propose phage P59 to be a new member of the family Herelleviridae.

A novel deep-sea bacteriophage possesses features of Wbeta-like viruses and prophages.

As the most abundant biological entities, viruses are major players in marine ecosystems. However, our knowledge about virus-host interactions and viral ecology in the deep sea remains very limited. In this study, a novel bacteriophage (designated as phage BVE2) infecting Bacillus cereus group bacteria, was isolated from deep-sea sediments. Phage BVE2 caused host lysis within 1.5 h after infection. However, the presence of two integrase-encoding genes in the BVE2 genome suggested that BVE2 may also follow a temperate strategy. The genome of phage BVE2 is approximately 20 kb in length and is predicted to encode 28 proteins. Genomic and phylogenetic analysis suggested that BVE2 is a highly mosaic phage that has inherited genetic features from Wbeta-like viruses, B. cereus prophages, and its host, suggesting that frequent horizontal gene transfer events occurred during its evolution. This study will help to reveal the evolutionary history of Wbeta-like viruses and improve our understanding of viral diversity and virus-host interactions in the deep sea.

Phage vB_BmeM-Goe8 infecting Bacillus megaterium DSM319.

vB_BmeM-Goe8 is a phage preying on Bacillus megaterium. Its genome has a GC content of 38.9%, is 161,583 bp in size, and has defined ends consisting of 7436-bp-long terminal repeats. It harbours 11 genes encoding tRNAs and 246 coding DNA sequences, 66 of which were annotated. The particle reveals Myoviridae morphology, and the formation of a double baseplate upon tail sheath contraction indicates morphological relatedness to the group of SPO1-like phages. BLASTn comparison against the NCBI non-redundant nucleotide database revealed that Bacillus phage Mater is the closest relative of vB_BmeM-Goe8.

Complete genome sequence of the novel phage vB_BthS-HD29phi infecting Bacillus thuringiensis.

The phage vB_BthS-HD29phi infecting Bacillus thuringiensis strain HD29 was isolated and purified. The morphology of the phage showed that it belongs to the family Siphoviridae. The phage genome was 32,181 bp in length, comprised linear double-stranded DNA with an average G + C content of 34.9%, and exhibited low similarity to known phage genomes. Genomic and phylogenetic analysis revealed that vB_BthS-HD29phi is a novel phage. In total, 50 putative ORFs were predicted in the phage genome, and only 18 ORFs encoded proteins with known functions. This article reports the genome sequence of a new tailed phage and increases the known genetic diversity of tailed phages.

Bacillus Phage vB_BtS_B83 Previously Designated as a Plasmid May Represent a New Siphoviridae Genus.

The Bacillus cereus group of bacteria includes, inter alia, the species known to be associated with human diseases and food poisoning. Here, we describe the Bacillus phage vB_BtS_B83 (abbreviated as B83) infecting the species of this group. Transmission electron microscopy (TEM) micrographs indicate that B83 belongs to the Siphoviridae family. B83 is a temperate phage using an arbitrium system for the regulation of the lysis-lysogeny switch, and is probably capable of forming a circular plasmid prophage. Comparative analysis shows that it has been previously sequenced, but was mistaken for a plasmid. B83 shares common genome organization and >46% of proteins with other the Bacillus phage, BMBtp14. Phylograms constructed using large terminase subunits and a pan-genome presence-absence matrix show that these phages form a clade distinct from the closest viruses. Based on the above, we propose the creation of a new genus named Bembunaquatrovirus that includes B83 and BMBtp14.

Genome sequence of Bacillus anthracis typing phage AP631.

AP631, a virulent bacteriophage of Bacillus anthracis, is widely used in China to identify anthrax bacteria. In this study, we report the complete AP631 phage genome sequence as well as comparative genomic analysis with other bacteriophages of B. cereus and related species. The double-stranded circular DNA genome of phage AP631 was 39,549 bp in length with 35.01% G + C content. The phage genome contained 56 putative protein-coding genes but no rRNA or tRNA genes. Comparative phylogenetic analysis of the phage major capsid proteins and terminase large subunits showed that phage AP631 belongs to the B. cereus sensu lato phage clade II. Comparative genomic analysis revealed a high degree of sequence similarity between phage AP631 and B. anthracis phages Wbeta, Gamma, Cherry, and Fah, as well as three AP631-specific genes bearing no significant similarity to those of other phages.

Complete nucleotide sequence analysis of a novel Bacillus subtilis-infecting phage, BSP38, possibly belonging to a new genus in the subfamily Spounavirinae.

Bacillus subtilis-infecting phage BSP38 was isolated from a sewage sample. Morphologically, BSP38 was found to be similar to members of the subfamily Spounavirinae, family Myoviridae. Its genome is 153,268 bp long with 41.8% G+C content and 254 putative open reading frames (ORFs) as well as six tRNAs. A distinguishing feature for this phage among the reported B. subtilis-infecting phages is the presence of an encoding ORF, putative tRNAHis guanylyltransferase-like protein. Genomic comparisons with the other reported phages strongly suggest that BSP38 should be considered a member of a new genus in the subfamily Spounavirinae.

Isolation of A Novel Bacillus thuringiensis Phage Representing A New Phage Lineage and Characterization of Its Endolysin.

Phages, the parasites of bacteria, are considered as a new kind of antimicrobial agent due to their ability to lyse pathogenic bacteria. Due to the increase of available phage isolates, the newly isolated phage showed increasing genomic similarities with previously isolated phages. In this study, the novel phage vB_BthS_BMBphi, infecting the Bacillus thuringiensis strain BMB171, is isolated and characterized together with its endolysin. This phage is the first tadpole-like phage infecting the Bacillus strains. Genomic analysis shows that the phage genome is dissimilar to all those of previously characterized phages, only exhibiting low similarities with partial regions of the B. thuringiensis prophages. Phylogenetic analysis revealed that the phage was distant from the other Bacillus phages in terms of evolution. The novel genome sequence, the distant evolutionary relationship, and the special virion morphology together suggest that the phage vB_BthS_BMBphi could be classified as a new phage lineage. The genome of the phage is found to contain a restriction modification system, which might endow the phage with immunity to the restriction modification system of the host bacterium. The function of the endolysin PlyBMB encoded by the phage vB_BthS_BMBphi was analyzed, and the endolysin could lyse all the tested Bacillus cereus group strains, suggesting that the endolysin might be used in controlling pathogenic B. cereus group strains. The findings of this study enrich the understanding of phage diversity and provide a resource for controlling the B. cereus group pathogenic bacteria.

Genomic Analysis of the Recent Viral Isolate vB_BthP-Goe4 Reveals Increased Diversity of φ29-Like Phages.

We present the recently isolated virus vB_BthP-Goe4 infecting Bacillus thuringiensis HD1. Morphological investigation via transmission electron microscopy revealed key characteristics of the genus Phi29virus, but with an elongated head resulting in larger virion particles of approximately 50 nm width and 120 nm height. Genome sequencing and analysis resulted in a linear phage chromosome of approximately 26 kb, harbouring 40 protein-encoding genes and a packaging RNA. Sequence comparison confirmed the relation to the Phi29virus genus and genomes of other related strains. A global average nucleotide identity analysis of all identified φ29-like viruses revealed the formation of several new groups previously not observed. The largest group includes Goe4 and may significantly expand the genus Phi29virus (Salasvirus) or the Picovirinae subfamily.

Genomic characterization of three novel Basilisk-like phages infecting Bacillus anthracis.

BACKGROUND: In the present study, we sequenced the complete genomes of three novel bacteriophages v_B-Bak1, v_B-Bak6, v_B-Bak10 previously isolated from historical anthrax burial sites in the South Caucasus country of Georgia. We report here major trends in the molecular evolution of these phages, which we designate as "Basilisk-Like-Phages" (BLPs), and illustrate patterns in their evolution, genomic plasticity and core genome architecture. RESULTS: Comparative whole genome sequence analysis revealed a close evolutionary relationship between our phages and two unclassified Bacillus cereus group phages, phage Basilisk, a broad host range phage (Grose JH et al., J Vir. 2014;88(20):11846-11860) and phage PBC4, a highly host-restricted phage and close relative of Basilisk (Na H. et al. FEMS Microbiol. letters. 2016;363(12)). Genome comparisons of phages v_B-Bak1, v_B-Bak6, and v_B-Bak10 revealed significant similarity in sequence, gene content, and synteny with both Basilisk and PBC4. Transmission electron microscopy (TEM) confirmed the three phages belong to the Siphoviridae family. In contrast to the broad host range of phage Basilisk and the single-strain specificity of PBC4, our three phages displayed host specificity for Bacillus anthracis. Bacillus species including Bacillus cereus, Bacillus subtilis, Bacillus anthracoides, and Bacillus megaterium were refractory to infection. CONCLUSIONS: Data reported here provide further insight into the shared genomic architecture, host range specificity, and molecular evolution of these rare B. cereus group phages. To date, the three phages represent the only known close relatives of the Basilisk and PBC4 phages and their shared genetic attributes and unique host specificity for B. anthracis provides additional insight into candidate host range determinants.

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40-Bacillus Specific Endolysins.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.

Complete Nucleotide Sequence Analysis of a Novel Bacillus subtilis-Infecting Bacteriophage BSP10 and Its Effect on Poly-Gamma-Glutamic Acid Degradation.

While the harmful effects of lactic acid bacterial bacteriophages in the dairy industry are well-established, the importance of Bacillus subtilis-infecting bacteriophages on soybean fermentation is poorly-studied. In this study, we isolated a B. subtilis-infecting bacteriophage BSP10 from Meju (a brick of dried fermented soybean) and further characterized it. This Myoviridae family bacteriophage exhibited a narrow host range against B. subtilis strains (17/52, 32.7%). The genome of bacteriophage BSP10 is 153,767 bp long with 236 open reading frames and 5 tRNAs. Comparative genomics (using dot plot, progressiveMauve alignment, heat-plot, and BLASTN) and phylogenetic analysis strongly suggest its incorporation as a new species in the Nit1virus genus. Furthermore, bacteriophage BSP10 was efficient in the growth inhibition of B. subtilis ATCC 15245 in liquid culture and in Cheonggukjang (a soybean fermented food) fermentation. Artificial contamination of as low as 10² PFU/g of bacteriophage BSP10 during Cheonggukjang fermentation significantly reduced bacterial numbers by up to 112 fold in comparison to the control (no bacteriophage). Moreover, for the first time, we experimentally proved that B. subtilis-infecting bacteriophage greatly enhanced poly-γ-glutamic acid degradation during soybean fermentation, which is likely to negatively affect the functionalities of Cheonggukjang.

Complete genome sequence of bacteriophage Deep-Purple, a novel member of the family Siphoviridae infecting Bacillus cereus.

Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60 °C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.

Identification and genomic comparison of temperate bacteriophages derived from emetic Bacillus cereus.

Cereulide-producing Bacillus cereus isolates can cause serious emetic (vomiting) syndrome and even acute lethality. As mobile genetic elements, the exploration of prophages derived from emetic B. cereus isolates will help in our understanding of the genetic diversity and evolution of these pathogens. In this study, five temperate phages derived from cereulide-producing B. cereus strains were induced, with four of them undergoing genomic sequencing. Sequencing revealed that they all belong to the Siphoviridae family, but presented in different forms in their hosts. PfNC7401 and PfIS075 have typical icosahedral heads, probably existing alone as phagemids in the host with self-replicating capability in the lysogenic state. PfEFR-4, PfEFR-5, and PfATCC7953 have elongated heads, with the genomes of the former two identified as linear dsDNA, which could be integrated into the host genome during the lysogenic state. Genomic comparison of the four phages with others also derived from emetic B. cereus isolates showed similar genome structures and core genes, thus displaying host spectrum specificity. In addition, phylogenic analysis based on the complete genome and conserved tail fiber proteins of 36 Bacillus species-derived phages confirmed that the phages derived from emetic B. cereus strains were highly similar. Furthermore, one endolysin LysPfEFR-4 was cloned and showed lytic activity against all tested emetic B. cereus strains and cross-lytic activity against some other pathogenic bacteria, implying a potential to control bacterial contamination in the food supply.