Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

Are kuravirus capsid diameters quantized? The first all-atom genome tracing method for double-stranded DNA viruses.

The revolution in cryo-electron microscopy has resulted in unprecedented power to resolve large macromolecular complexes including viruses. Many methods exist to explain density corresponding to proteins and thus entire protein capsids have been solved at the all-atom level. However methods for nucleic acids lag behind, and no all-atom viral double-stranded DNA genomes have been published at all. We here present a method which exploits the spiral winding patterns of DNA in icosahedral capsids. The method quickly generates shells of DNA wound in user-specified, idealized spherical or cylindrical spirals. For transition regions, the method allows guided semiflexible fitting. For the kuravirus SU10, our method explains most of the density in a semiautomated fashion. The results suggest rules for DNA turns in the end caps under which two discrete parameters determine the capsid inner diameter. We suggest that other kuraviruses viruses may follow the same winding scheme, producing a discrete rather than continuous spectrum of capsid inner diameters. Our software may be used to explain the published density maps of other double-stranded DNA viruses and uncover their genome packaging principles.

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.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Structural Study of the Cobetia marina Bacteriophage 1 (Carin-1) by Cryo-EM.

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Quorum sensing positively regulates CPS-dependent Autographiviridae phage infection in Vibrio alginolyticus.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

Phage-resistant Streptomyces abietis and its telomycin bioactive metabolite as a possible alternative to antibiotics.

Multidrug-resistant pathogens are now thought to be the primary global causes of disease and death. Therefore, it is imperative to develop new effective bioactive compounds from microbial sources, such as Streptomyces species. Nevertheless, the pharmaceutical industry suffered financial losses and low-quality end products as a result of Streptomyces bacteriophage contamination. To reduce the likelihood of phage-induced issues in the medical industry, it is crucial to develop a method for finding phage-resistant strains. Hence, we aimed to isolate and characterize Streptomyces spp. and Streptomyces phages from various rhizospheric soil samples in Egypt and to investigate their antibacterial activities. Moreover, we targeted development of a Streptomyces phage-resistant strain to extract its active metabolites and further testing its antibacterial activity. Herein, the antibacterial activities of the isolated 58 Streptomyces isolates showed that 10 (17.2 %) Streptomyces isolates had antibacterial activities against the tested bacteria including Listeria monocytogenes, E. coli O157, Acinetobacter baumannii, methicillin resistant-vancomycin-intermediate Staphylococcus aureus (MRSA-VISA) and Micrococcus luteus. Three lytic bacteriophages (ϕPRSC1, ϕPRSC2, and ϕPRSC4) belonging to the families Siphoviridae and Podoviridae were obtained from the rhizospheric soil samples using the most potent S. abietis isolate as the host strain. The three isolated Streptomyces phages were thermostable, ultraviolet stable, infectious, and had a wide range of hosts against the 10 tested Streptomyces isolates with antibacterial activities. The DNA of the ϕPRSC1 and ϕPRSC4 phages were resistant to digestion by EcoRI and HindIII, but the DNA of ϕPRSC2 was resistant to digestion by EcoRI and sensitive to digestion by HindIII. Of note, we developed a S. abietis strain resistant to the three isolated phages and its antibacterial activities were twice that of the wild strain. Finally, telomycin was recognized as an antibacterial metabolite extracted from phage-resistant S. abietis strain, which was potent against the tested Gram-positive bacteria including L. monocytogenes, MRSA-VISA, and M. luteus. Thus, our findings open new horizons for researching substitute antimicrobial medications for both existing and reemerging illnesses.

Isolation and characterization of a relatively broad-spectrum phage against Escherichia coli.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. In this study, PH444, a relatively broad-spectrum obligate lytic phage, was screened from 48 Shiga toxin-producing Escherichia coli (STEC) phages isolated from farm manure samples and sewage samples in order to conduct genome-wide analysis, biological characterization, and a bacterial challenge experiment in milk. The results demonstrated that PH444 was a T7-like phage with a double-stranded DNA of 115,111 bp that belongs to the Kuravirus and was stable at temperatures between 4 and 50 °C and a pH range of 3 to 11. After adding PH444, the bacterial load in milk could be reduced from 3 × 103 PFU/ mL to zero within 1 h. In consideration of the biological properties of phage PH444, it was, therefore, demonstrated that PH444 has the potential to be used in phage biocontrol.

Characterization of selected phages for biocontrol of food-spoilage pseudomonads.

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

Biological characterization and genomic analysis of a novel bacteriophage, MopsHU1, infecting Morganella psychrotolerans.

Morganella psychrotolerans is a histamine-producing bacterium that causes histamine poisoning. In this study, we isolated and characterized a novel phage, MopsHU1, that infects M. psychrotolerans. MopsHU1 is a podovirus with a limited host spectrum. Genomic analysis showed that MopsHU1 belongs to the family Autographiviridae, subfamily Studiervirinae, and genus Kayfunavirus. Comparative analysis revealed that the MopsHU1 genome is similar to those of Citrobacter phage SH3 and Cronobacter phage Dev2. Moreover, the Escherichia coli phage K1F genome is also similar, except for its tailspike gene sequence. These results expand our understanding of the Kayfunavirus phages that infect Morganella spp. Note: The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank database under the accession number LC799501.

Isolation and Biological Characteristics of a Novel Phage and Its Application to Control Vibrio Parahaemolyticus in Shellfish Meat.

Vibrio parahaemolyticus is a common foodborne pathogenic bacterium. With the overuse of antibiotics, an increasing proportion of drug-resistant strains are emerging, which puts enormous pressure on public health. In this study, a V. parahaemolyticus-specific phage, VP41s3, was isolated. The head length, width, and tail length of the phage were 77.7 nm, 72.2 nm, and 17.5 nm, respectively. It remained active in the temperature range of 30-50°C and pH range of 4-11. The lytic curve of phage VP41s3 showed that the host bacteria did not grow until 11 h under phage treatment at MOI of 1000, indicating that the phage had good bacteriostatic ability. When it was added to shellfish contaminated with V. parahaemolyticus (15°C, 48 h), the number of bacteria in the experimental group was 2.11 log10 CFU/mL lower than that in the control group at 24 h. Furthermore, genomic characterization and phylogenetic analysis indicated that phage VP41s3 was a new member of the Podoviridae family. The genome contained 50 open reading frames (ORFs), in which the ORF19 (thymidine kinase) was an enzyme involved in the pyrimidine salvage pathway, which might lead to the accelerated DNA synthesis efficiency after phage entered into host cells. This study not only contributed to the improvement of phage database and the development of beneficial phage resources but also revealed the potential application of phage VP41s3 in food hygiene and safety.

Complete genome sequence analysis, morphology and structural protein identification of two Bacillus subtilis phages, BSTP4 and BSTP6, which may form a new species in the genus Salasvirus.

In the present study, two new Bacillus subtilis phages, BSTP4 and BSTP6, were isolated and studied further. Morphologically, BSTP4 and BSTP6 are podoviruses with complete genome of 19,145 (39.9% G + C content) and 19,367 bp (39.8% G + C content), respectively, which became among the smallest Bacillus phages. Three most prominent structural proteins were separated and identified as pre-neck appendage, major head, and head fiber proteins using LC-MS/MS. Both phages encode putative terminal proteins (TP) and contain short inverted terminal repeats (ITRs) which may be important for their replication. In addition, non-coding RNA (pRNA) and parS sites were identified which may be required for DNA packaging and their maintenance inside the host, respectively. Furthermore, the phage genome sequences show significant similarity to B. subtilis group species genome sequences. Finally, phylogenomic and phylogenetic analyses suggest that BSTP4 and BSTP6 may form a new species in the genus Salasvirus, subfamily Picovirinae of family Salasmaviridae. Considering the small numbers of ICTV-accepted B. subtilis phages and the importance of the host in the food industry and biotechnology, the current study helps to improve our understanding of the diversity of B. subtilis phages and shed light on the phage-host relationships.

Whole genome sequencing and annotation of a lysogenic phage vB_EcoP_DE5 isolated from donkey-derived Escherichia coli.

A lysogenic phage vB_EcoP_DE5 (hereafter designated DE5) was isolated from donkey-derived Escherichia coli. The bacteriophage was examined by transmission electron microscopy, and the result showed that DE5 belonged to the genus Kuravirus. DE5 was sensitive to changes in temperature and pH, and it could maintain its activity at pH 7 and below 60 â„ƒ. The whole genome sequencing revealed that DE5 had a double-stranded DNA genome of 77, 305 bp with 42.09% G+C content. A total of 126 open reading frames (ORFs) were identified, including functional genes related to phage integration, DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. One phage integrase gene, one autotransporter adhesin gene, and one tRNA gene were predicted in the whole genome, and no genes associated with drug resistance were identified. The phage DE5 integrase contained 187 amino acids and belonged to the small serine recombinase family. BLASTn analysis revealed that phage DE5 had a high-sequence identity (96%) with E. coli phage SU10. Phylogenetic analysis showed that phage DE5 was a member of the genus Kuravirus. The whole genome sequencing of lysogenic phage DE5 enhanced our understanding of lysogenic phages and their therapeutic applications.

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8.

All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3'-5' exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

Isolation and Characterization of the First Zobellviridae Family Bacteriophage Infecting Klebsiella pneumoniae.

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage's ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly.

Phage Targeting Neonatal Meningitis E. coli K1 In Vitro in the Intestinal Microbiota of Pregnant Donors and Impact on Bacterial Populations.

Escherichia coli K1 is a leading cause of neonatal meningitis. The asymptomatic carriage of these strains in the maternal intestinal microbiota constitutes a risk of vertical transmission to the infant at birth. The aim of this work was to evaluate the efficacy of phage therapy against E. coli K1 in an intestinal environment and its impact on the intestinal microbiota. For this purpose, three independent experiments were conducted on the SHIME® system, the first one with only the phage vB_EcoP_K1_ULINTec4, the second experiment with only E. coli K1 and the last experiment with both E. coli K1 and the phage. Microbiota monitoring was performed using metagenetics, qPCR, SCFA analysis and the induction of AhR. The results showed that phage vB_EcoP_K1_ULINTec4, inoculated alone, was progressively cleared by the system and replicates in the presence of its host. E. coli K1 persisted in the microbiota but decreased in the presence of the phage. The impact on the microbiota was revealed to be donor dependent, and the bacterial populations were not dramatically affected by vB_K1_ULINTec4, either alone or with its host. In conclusion, these experiments showed that the phage was able to infect the E. coli K1 in the system but did not completely eliminate the bacterial load.

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection.

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed.

Metagenomic analysis of wastewater phageome from a University Hospital in Turkey.

Phage DNA analysis gives opportunity to understand living ecosystem of the environment where the samples are taken. In the present study, we analyzed phage DNA obtained from wastewater sample of university hospital sewage. After filtration, long high-speed centrifugation was done to collect phages. DNA was extracted from pellet by phenol chloroform extraction and used for NGS sequencing. The host profile, taxonomic and functional analyses were performed using MG-RAST, and ResFinder program was used for resistance gene detection. High amounts of reads belong to bacteriophage groups (~ 95%) from our DNA sample were obtained and all bacteriophage reads were found belonging to Caudovirales order and Myoviridae (56%), Siphoviridae (43%), and Podoviridae (0.02%) families. The most common host genera were Escherichia (88.20%), Salmonella (5.49%) and Staphylococcus (5.19%). SEED subsystems hits were mostly structural parts and KEGG Orthology hits were nucleotide- and carbohydrate metabolism-related genes. No anti-microbial resistance genes were detected. Our bacteriophage DNA purification method is favorable for phage metagenomic studies. Dominance of coliphages may explain infrequent Podoviridae. Dominancy of structural genes and auxiliary genes is probably due to abundance of lytic phages in our sample. Absence of antibiotic resistance genes even in hospital environment phages indicates that phages are not important carrier of resistance genes.

Nettle manure: an unsuspected source of bacteriophages active against various phytopathogenic bacteria.

Screening of 10 environmental samples (mainly of rhizospheric origin) for lytic activity against two bacterial phytopathogens, Pseudomonas syringae pv. tomato DC3000 (CFBP2212) and Xanthomonas hortorum pv. vitians (CFBP3979), revealed that four samples harboured phages that were active against one strain. Only one sample, composed of an artisanal nettle liquid manure, contained phages able to lyse both strains. Electron microscopy revealed the presence of tailed bacteriophages, with all phages isolated on the Xanthomonas strain displaying a contractile tail typical of members of the family Myoviridae, whereas phages isolated on the Pseudomonas strain were related to members of the family Siphoviridae and short-tailed members of the family Podoviridae. Sequence analysis of the two Podoviridae-like bacteriophages isolated on Pseudomonas syringae pv. tomato, Pst_GM1 isolated from nettle manure and Pst_GIL1 isolated from infected lettuce leaves, revealed (i) strong homology between the two isolated phages, (ii) a high degree of sequence similarity to various phages isolated from various environments and from different geographical locations, and (iii) similarity of these phages to members of the family Autographiviridae, and more precisely, the genus Ghunavirus. Further investigation of the potential of nettle manure to host phages that could be active against a wider range of strains revealed that it contained phages active against 10 phytopathogens (out of 16 tested). Thus, nettle manure (and likely other plant manures) could represent a valuable source of phages, especially those targeting bacterial phytopathogens, in the same way that anthropized environments such as sewage are widely used as sources of phages active against opportunistic or acute pathogens of humans.

Genomic characterization of bacteriophage pSal-SNUABM-01, a novel elongated-head phage infecting Salmonella sp.

Salmonellosis is a disease of critical concern for public health, and the use of bacteriophages is among the most promising approaches to combating Salmonella. As Salmonella has various serotypes and strains, and bacteriophages are virulent to specific hosts, it is important to isolate phages and evaluate interactions with their hosts. In the present study, a novel Salmonella-infecting bacteriophage, pSal-SNUABM-01, was isolated and characterized. Transmission electron microscopy revealed that the bacteriophage is a member of the family Podoviridae and possesses an elongated head and a short tail. The phage genome is circular and 89,500 bp in size. A total of 162 open reading frames were predicted, eight of which were tRNAs. Morphological and genomic analysis revealed that pSal-SNUABM-01 is closely related to phage 7-11. In phylogenetic analysis, pSal-SNUABM-01 and 7-11 did not cluster together with the members of any established genus, suggesting that these two phages comprise a novel genus. The results of this study enhance our understanding of the phylogeny of the family Podoviridae and might be applicable to the development of bacteriophage treatments against Salmonella infections.