Acinetobacter baumannii satellite phage Aci01-2-Phanie depends on a helper myophage for its multiplication.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

Biological properties of Staphylococcus virus ΦSA012 for phage therapy.

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

Structural Studies of the Phage G Tail Demonstrate an Atypical Tail Contraction.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host-phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head-neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head-neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G's sheath structure in the non-contracted and contracted states are similar to phage T4's sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath's helical symmetry, the sheath protein's evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head-neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Comparative Genomics of Three Novel Jumbo Bacteriophages Infecting Staphylococcus aureus.

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is ∼269 kb with each genome encoding ∼263 predicted protein-coding genes. Phage genome organization and content are similar to those of known jumbo phages of Bacillus sp., including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete virion and nonvirion RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates. IMPORTANCE This study describes the comparative genomics of the following three novel S. aureus jumbo phages: MarsHill, Madawaska, and Machias. These three S. aureus myophages represent an emerging class of S. aureus phage. These genomes contain abundant introns which show a pattern consistent with repeated acquisition rather than vertical inheritance, suggesting intron acquisition and loss are active processes in the evolution of these phages. These phages have presumably hypermodified DNA which inhibits sequencing by several different common platforms. Therefore, these phages also represent potential genomic diversity that has been missed due to the limitations of standard sequencing techniques. In particular, such hypermodified genomes may be missed by metagenomic studies due to their resistance to standard sequencing techniques. Phage MarsHill was found to be able to transduce host DNA at levels comparable to that found for other transducing S. aureus phages, making it a potential vector for horizontal gene transfer in the environment.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523.

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

Computational image analysis of the baseplate-tail complex of O1 ElTor vibriophage M4.

We performed an in-depth computational image analysis of the baseplate-tail complex of the M4 vibriophage and identified seven major densities in its baseplate, which notably share structural similarities with baseplate modules of a number of other bacteriophages belonging to different species. Employing computational analysis, we explained the helical organization of the sheath protein, wrapping the tail tube. Based on the results obtained in this work along with the proteomics information published previously, we are able to decipher the plausible roles assigned to the different components of the M4 baseplate during infection of the host.

3D structure of three jumbo phage heads.

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to Myoviridae (ΦRP13; T=21) was discovered as well as two others, which are more common for jumbo phages (T=27 and T=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.

Biological characteristics and whole-genome analysis of the Enterococcus faecalis phage PEf771.

Enterococcus faecalis is a common pathogen causing refractory periapical periodontitis and secondary intraradicular infections. In this study, E. faecalis YN771 isolated from a re-treated root canal at a stomatology department was used as the host bacterium and was co-cultured with wastewater from the same department and patient samples to isolate a phage that lyses E. faecalis. We studied the biological and genomic characteristics of this phage. Transmission electron microscopy showed that this phage's head is icosahedral in structure, with a head diameter of around 98.4 nm, and a contractile tail of around 228.5 nm in length and a diameter of 17.3 nm. The phage was identified as a member of the Myoviridae family and named PEf771. It is sensitive to proteinase K but resistant to chloroform and Triton X-100. Its lytic cycle is 45 min, burst size is 78, optimal multiplicity of infection is 0.1, lysis spectrum is narrow, and host strain specificity is strong. Its optimal growth temperature is 37 °C, most suitable pH is 6.0, and is sensitive to ultraviolet radiation. Whole-genome sequencing of PEf771 indicated it has a genome size of 151 052 bp, with a GC content of 36.97%, and encodes 197 proteins plus 26 tRNAs. PEf771 is most closely related to E. faecalis phage EFDG1. Phage PEf771 has strong host specificity and lytic ability, so it is important to further characterize this phage and its interaction with E. faecalis.

Isolation and complete genome sequence of a novel cyanophage, S-B05, infecting an estuarine Synechococcus strain: insights into environmental adaptation.

A new cyanophage, S-B05, infecting a phycoerythrin-enriched (PE-type) Synechococcus strain was isolated by the liquid infection method, and its morphology and genetic features were examined. Phylogenetic analysis and morphological observation confirmed that S-B05 belongs to the family Myoviridae of the order Caudovirales. Its genome was fully sequenced, and found to be 208,857 bp in length with a G + C content of 39.9%. It contained 280 potential open reading frames and 123 conserved domains. Ninety-eight functional genes responsible for cyanophage structuring and packaging, DNA replication and regulation, and photosynthesis were identified, as well as genes encoding 172 hypothetical proteins. The genome of S-B05 is most similar to that of Prochlorococcus phage P-TIM68. Homologues of open reading frames of S-B05 can be found in various marine environments, as revealed by comparison of the S-B05 genome sequence to sequences in marine viral metagenomic databases. The presence of auxiliary metabolic genes (AMGs) related to photosynthesis, carbon metabolism, and phosphorus assimilation, as well as the phylogenetic relationships based on AMGs and the complete genome sequence, reflect the phage-host interaction mechanism or the specific adaptation strategy of the host to environmental conditions. The genome sequence information reported here will provide an important basis for further study of the adaptive evolution and ecological role of cyanophages and their hosts in the marine environment.

Isolation and characterization of Hena1 - a novel Erwinia amylovora bacteriophage.

Fire blight, caused by plant pathogenic bacterium Erwinia amylovora, is one of the most important diseases of Rosaceae plants. Due to the lack of effective control measures, fire blight infections pose a recurrent threat on agricultural production worldwide. Recently, bacterial viruses, or bacteriophages, have been proposed as environmentally friendly natural antimicrobial agents for fire blight control. Here, we isolated a novel bacteriophage Hena1 with activity against E. amylovora. Further analysis revealed that Hena1 is a narrow-host-range lytic phage belonging to Myoviridae family. Its genome consists of a linear 148,842 bp dsDNA (48.42% GC content) encoding 240 ORFs and 23 tRNA genes. Based on virion structure and genomic composition, Hena1 was classified as a new species of bacteriophage subfamily Vequintavirinae. The comprehensive analysis of Hena1 genome may provide further insights into evolution of bacteriophages infecting plant pathogenic bacteria.

Characterization of a bacteriophage, vB_Eco4M-7, that effectively infects many Escherichia coli O157 strains.

The characterization of a recently isolated bacteriophage, vB_Eco4M-7, which effectively infects many, though not all, Escherichia coli O157 strains, is presented. The genome of this phage comprises double-stranded DNA, 68,084 bp in length, with a GC content of 46.2%. It contains 96 putative open reading frames (ORFs). Among them, the putative functions of only 35 ORFs were predicted (36.5%), whereas 61 ORFs (63.5%) were classified as hypothetical proteins. The genome of phage vB_Eco4M-7 does not contain genes coding for integrase, recombinase, repressors or excisionase, which are the main markers of temperate viruses. Therefore, we conclude that phage vB_Eco4M-7 should be considered a lytic virus. This was confirmed by monitoring phage lytic development by a one-step growth experiment. Moreover, the phage forms relatively small uniform plaques (1 mm diameter) with no properties of lysogenization. Electron microscopic analyses indicated that vB_Eco4M-7 belongs to the Myoviridae family. Based on mass spectrometric analyses, including the fragmentation pattern of unique peptides, 33 phage vB_Eco4M-7 proteins were assigned to annotated open reading frames. Importantly, genome analysis suggested that this E. coli phage is free of toxins and other virulence factors. In addition, a similar, previously reported but uncharacterized bacteriophage, ECML-117, was also investigated, and this phage exhibited properties similar to vB_Eco4M-7. Our results indicate that both studied phages are potential candidates for phage therapy and/or food protection against Shiga toxin-producing E. coli, as the majority of these strains belong to the O157 serotype.

Phage vB_BveM-Goe7 represents a new genus in the subfamily Bastillevirinae.

Bacillus velezensis FZB42 is a Gram-positive, endospore-forming rhizobacterium that is associated with plant roots and promotes plant growth. It was used as host to isolate phage vB_BveM-Goe7 (Goe7). Goe7 exhibits a Myoviridae morphology with a contractile tail and an icosahedral head. Its genome is 158,674 bp in size and contains 5137-bp-long terminal repeats (LTRs). It also contains five tRNA-encoding genes and 251 coding DNA sequences (CDS), of which 65 were annotated. The adsorption constant of Goe7 is 6.1 ± 0.24 × 10-8 ml/min, with a latency period of 75 min and a burst size of 114 particles per burst. A BLASTn sequence comparison against the non-redundant nucleotide database of NCBI revealed that Goe7 is most similar to Bacillus subtilis phage vB_BsuM-Goe3.

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.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

A cornucopia of Shigella phages from the Cornhusker State.

Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.

Morphologic and genomic characterization of a broad host range Salmonella enterica serovar Pullorum lytic phage vB_SPuM_SP116.

For effective use of phages as antimicrobial agents for controlling multidrug resistant S. Pullorum, it is important to understand phage biology. A lytic S. Pullorum phage was isolated and characterized from chicken feces, and its whole genome was sequenced and analyzed. A new lytic phage-vB_SPuM_SP116 (in brief SP116)- isolated and characterized using S. Pullorum SPu-116 as its host belongs to Myoviridae A1 group. Phage SP116 had a lytic effect on 27 of 37 (72.9%) different serotypes of clinical Salmonella strains. It showed a high bactericidal activity in killing all pathogens in cultures containing 5 × 105 cfu/mL and achieved more than 6.58 and 5.97 log unit reductions in cultures containing 5 × 106 cfu/mL and 5 × 107 cfu/mL, respectively. The one-step growth curve showed that the burst size was up to 118 pfu/bacterial cell. Complete genome sequence analysis revealed a linear, double-stranded DNA genome of 87,510 bp with an average G + C content of 38.84%, including 128 predicted open reading frames (ORFs) and 22 tRNA genes. SP116 was classified as a Felix O1 virus based upon the general phage characterization and the genomic information. Regarding its high efficacy in preventing especially S. Pullorum infection and its lack of any bacterial virulence, antimicrobial resistance, and lysogenesis genes, it could be a potential alternative candidate for the treatment of S. Pullorum infections.

Green approach to phytopathogen: Characterization of lytic bacteriophages of Pseudomonas sp., an etiology of the bacterial blight of pomegranate.

Two morphologically different bacteriophages were isolated from the river and soil samples from various locations of Maharashtra, India against the phytopathogen Pseudomonas sp. that was recently reported to cause a new bacterial blight of pomegranate. Both the phages belonged to the order Caudovirales representing the families Siphoviridae (vB_Psp.S_PRɸL2) and Myoviridae (vB_Psp.M_SSɸL8). The multiplicity of infection ranged from 0.01 to 0.1, phage adsorption rate from 39% to 66%, latent period from 10 to 20 min with a burst size of 24-85 phage particles per infected host cell. The genome size of phages PRɸL2 and SSɸL8 was approximately 25.403 kb and 29.877 kb respectively. Restriction digestion pattern of phage genomic DNA was carried out for phage PRɸL2, Eco RI resulted in two bands and Hind III resulted in three bands while for phage SSɸL8, both Eco RI and Hind III each resulted in three bands. SDS-PAGE protein profile showed six bands for PRɸL2 and nine bands for SSɸL8 of different proteins. Phages showed high pH stability over a range of 4-9, temperature stability over a range of 4-50 °C and UV radiation showed a reduction up to 89.36% for PRɸL2 and 96% for SSɸL8. In short, the present research work discusses for the first time in-detailed characterization of phages of a phytopathogen Pseudomonas sp. from Maharashtra, India, which can be further efficiently used for biological control of the causative agent of a new bacterial blight disease of pomegranate.

Characterization of SE-P3, P16, P37, and P47 bacteriophages infecting Salmonella Enteritidis.

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10-7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.

Expanding the Diversity of Myoviridae Phages Infecting Lactobacillus plantarum-A Novel Lineage of Lactobacillus Phages Comprising Five New Members.

Lactobacillus plantarum is a bacterium with probiotic properties and promising applications in the food industry and agriculture. So far, bacteriophages of this bacterium have been moderately addressed. We examined the diversity of five new L. plantarum phages via whole genome shotgun sequencing and in silico protein predictions. Moreover, we looked into their phylogeny and their potential genomic similarities to other complete phage genome records through extensive nucleotide and protein comparisons. These analyses revealed a high degree of similarity among the five phages, which extended to the vast majority of predicted virion-associated proteins. Based on these, we selected one of the phages as a representative and performed transmission electron microscopy and structural protein sequencing tests. Overall, the results suggested that the five phages belong to the family Myoviridae, they have a long genome of 137,973-141,344 bp, a G/C content of 36.3-36.6% that is quite distinct from their host's, and surprisingly, 7 to 15 tRNAs. Only an average 41/174 of their predicted genes were assigned a function. The comparative analyses unraveled considerable genetic diversity for the five L. plantarum phages in this study. Hence, the new genus "Semelevirus" was proposed, comprising exclusively of the five phages. This novel lineage of Lactobacillus phages provides further insight into the genetic heterogeneity of phages infecting Lactobacillus sp. The five new Lactobacillus phages have potential value for the development of more robust starters through, for example, the selection of mutants insensitive to phage infections. The five phages could also form part of phage cocktails, which producers would apply in different stages of L. plantarum fermentations in order to create a range of organoleptic outputs.

Characterization of the virome of Paracoccus spp. (Alphaproteobacteria) by combined in silico and in vivo approaches.

Bacteria of the genus Paracoccus inhabit various pristine and anthropologically-shaped environments. Many Paracoccus spp. have biotechnological value and several are opportunistic human pathogens. Despite extensive knowledge of their metabolic potential and genome architecture, little is known about viruses of Paracoccus spp. So far, only three active phages infecting these bacteria have been identified. In this study, 16 Paracoccus strains were screened for the presence of active temperate phages, which resulted in the identification of five novel viruses. Mitomycin C-induced prophages were isolated, visualized and their genomes sequenced and thoroughly analyzed, including functional validation of their toxin-antitoxin systems. This led to the identification of the first active Myoviridae phage in Paracoccus spp. and four novel Siphoviridae phages. In addition, another 53 prophages were distinguished in silico within genomic sequences of Paracoccus spp. available in public databases. Thus, the Paracoccus virome was defined as being composed of 66 (pro)phages. Comparative analyses revealed the diversity and mosaicism of the (pro)phage genomes. Moreover, similarity networking analysis highlighted the uniqueness of Paracoccus (pro)phages among known bacterial viruses.