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.

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.

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.

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.

Characterization and genome analysis of Pseudomonas aeruginosa phage vB_PaeP_Lx18 and the antibacterial activity of its lysozyme.

A lytic Pseudomonas aeruginosa phage, vB_PaeP_Lx18 (Lx18), was isolated from the sewage of a dairy farm. Biological characterization revealed that Lx18 was stable from 40 °C to 60 °C and over a wide range of pH values from 4 to 10. It was able to lyse 63.6% (21/33) of the P. aeruginosa strains tested and was able to reduce and disperse biofilms, with a biofilm reduction rate of 76.8%. Whole-genome sequencing showed that Lx18 is a dsDNA virus with a genome of 42,735 bp and G+C content of 62.16%. The genome contains 54 open reading frames (ORFs), 28 of which have known functions, including DNA replication and modification, transcriptional regulation, structural and packaging proteins, and host cell lysis. No virulence or tRNA genes were identified. Phylogenetic analysis showed that phage Lx18 belongs to the genus Phikmvvirus. The lysozyme of Lx18, Lys18, was cloned and expressed. The combined action of Lys18 and ethylenediaminetetraacetic acid (EDTA) had antibacterial activity against Pseudomonas aeruginosa. The study of phage Lx18 and its lysozyme will provide basic information for further research on the treatment of Pseudomonas aeruginosa infections.

Characterization and genome analysis of two new Aeromonas hydrophila phages, PZL-Ah1and PZL-Ah8.

Aeromonas hydrophila (A. hydrophila) is an opportunistic pathogen of fish, humans, and livestock, and has a severe negative impact on aquaculture development. Phage therapy is considered an alternative strategy for controlling bacterial infections and contamination. In this study, we isolated and characterized the genomes of two A. hydrophila-specific phages, PZL-Ah1 and PZL-Ah8, which, based on transmission electron microscopy, were identified as members of the family Podoviridae. Both of these phages had a relatively narrow host range, with lytic activity against Aeromonas spp. strains. Whole-genome sequence analysis revealed that PZL-Ah1 and PZL-Ah8 have a double-stranded DNA genome of 38,641 bp and 40,855 bp in length, with a GC content of 53.68% and 51.89%, respectively. Forty-four open reading frames (ORFs) were predicted in PZL-Ah1, and 52 were predicted in PZL-Ah8. Twenty-eight (63.6%) ORFs in PZL-Ah1 and 29 (55.8%) ORFs in PZL-Ah8 were predicted to encode functional proteins with homologs in the NCBI database, while the remaining ORFs were classified as encoding hypothetical proteins with unknown functions. A comparison with known phage genes suggested that ORF 02, ORF 29, and ORF 04 of PZL-Ah1 and ORF 2 and ORF 4 of PZL-Ah8 are involved in host cell lysis. This study expands the phage genome database and provides good candidates for phage typing applications.

S2B, a Temperate Bacteriophage That Infects Caulobacter Crescentus Strain CB15.

The Caulobacter crescentus strain CB15 has been the basis of numerous studies designed to characterize the biphasic life cycle of this bacterium. Here we describe a newly isolated podovirus, designated S2B, which is capable of integrating into the CB15 chromosome by recombining with the 3'-end of a particular tRNA-ser gene. In addition, we show that S2B is a representative of a family of closely related prophages that are present in the genomes of characterized strains from several Alphaproteobacteria genera. In contrast, only distantly related bacteriophage genomes are present in the GenBank database. The 42,846 bp S2B genome includes 262 bp terminal repeats, and it contains 62 genes of which 45 code for proteins of unknown function. Proteins with predicted functions include a T7 DNA polymerase, a T3/T7 RNA polymerase, and a T7 helicase/primase suggesting that S2B is part of the Studiervirinae subfamily of the Autographiviridae family.

Characterization and Complete Genomic Analysis of Vibrio Parahaemolyticus-Infecting Phage KIT05.

Vibrio parahaemolyticus is a bacterial pathogen in marine aquaculture systems and a major cause of food-borne illnesses worldwide. In the present study, Vibrio phage KIT05 was isolated from water collected from a shrimp farm in the Mekong Delta, Vietnam. It was characterized based on its morphology, growth curve, lytic properties, and genome sequence. Under the electron microscope, KIT05 particles had an icosahedral head with a diameter of 62.3 nm and a short tail of 24.1 nm. The one-step growth curve of KIT05 showed that its latency time was approximately 40 min and burst size was 18 plaque-forming units/cell. The genome of KIT05 comprises 50,628 bp with a GC content of 41.63%. It contains 60 open reading frames that are encoded within both strands and four tRNAs. The presence of direct terminal repeats of 130 bp at both ends of the KIT05 DNA was determined. According to phage morphology, genomic organization, and phylogeny analysis, Vibrio phage KIT05 was classified into the family Podoviridae. The genome annotation revealed that KIT05 had no virulent or lysogenic genes. This study may help identify a novel candidate for developing biocontrol agents for Vibrio parahaemolyticus.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Equine Intestinal O-Seroconverting Temperate Coliphage Hf4s: Genomic and Biological Characterization.

Tailed bacteriophages constitute the bulk of the intestinal viromes of vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are dominated mostly by temperate phages, while in horse feces virulent phages are more prevalent. To our knowledge, all the previously reported isolates of horse fecal coliphages are virulent. Temperate coliphage Hf4s was isolated from horse feces, from the indigenous equine Escherichia coli 4s strain. It is a podovirus related to the Lederbergvirus genus (including the well-characterized Salmonella bacteriophage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by the same bacteriophage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. IMPORTANCE The relationships between virulent and temperate bacteriophages and their impact on high-density symbiotic microbial ecosystems of animals are not always clear and may vary between species or even between individuals. The horse intestinal virome is dominated by virulent phages, and Hf4s is the first temperate equine intestinal coliphage characterized. It recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. These findings raise questions on the significance of bacteriophage-bacteriophage interactions within the ecology of microbial viruses in mammal intestinal ecosystems.