DEPhT: a novel approach for efficient prophage discovery and precise extraction.

Advances in genome sequencing have produced hundreds of thousands of bacterial genome sequences, many of which have integrated prophages derived from temperate bacteriophages. These prophages play key roles by influencing bacterial metabolism, pathogenicity, antibiotic resistance, and defense against viral attack. However, they vary considerably even among related bacterial strains, and they are challenging to identify computationally and to extract precisely for comparative genomic analyses. Here, we describe DEPhT, a multimodal tool for prophage discovery and extraction. It has three run modes that facilitate rapid screening of large numbers of bacterial genomes, precise extraction of prophage sequences, and prophage annotation. DEPhT uses genomic architectural features that discriminate between phage and bacterial sequences for efficient prophage discovery, and targeted homology searches for precise prophage extraction. DEPhT is designed for prophage discovery in Mycobacterium genomes but can be adapted broadly to other bacteria. We deploy DEPhT to demonstrate that prophages are prevalent in Mycobacterium strains but are absent not only from the few well-characterized Mycobacterium tuberculosis strains, but also are absent from all ∼30 000 sequenced M. tuberculosis strains.

Genome diversity of domesticated Acinetobacter baumannii ATCC 19606T strains.

Acinetobacter baumannii has emerged as an important opportunistic pathogen worldwide, being responsible for large outbreaks for nosocomial infections, primarily in intensive care units. A. baumannii ATCC 19606T is the species type strain, and a reference organism in many laboratories due to its low virulence, amenability to genetic manipulation and extensive antibiotic susceptibility. We wondered if frequent propagation of A. baumannii ATCC 19606T in different laboratories may have driven micro- and macro-evolutionary events that could determine inter-laboratory differences of genome-based data. By combining Illumina MiSeq, MinION and Sanger technologies, we generated a high-quality whole-genome sequence of A. baumannii ATCC 19606T, then performed a comparative genome analysis between A. baumannii ATCC 19606T strains from several research laboratories and a reference collection. Differences between publicly available ATCC 19606T genome sequences were observed, including SNPs, macro- and micro-deletions, and the uneven presence of a 52 kb prophage belonging to genus Vieuvirus. Two plasmids, pMAC and p1ATCC19606, were invariably detected in all tested strains. The presence of a putative replicase, a replication origin containing four 22-mer direct repeats, and a toxin-antitoxin system implicated in plasmid stability were predicted by in silico analysis of p1ATCC19606, and experimentally confirmed. This work refines the sequence, structure and functional annotation of the A. baumannii ATCC 19606T genome, and highlights some remarkable differences between domesticated strains, likely resulting from genetic drift.

Complete genome sequence of the newly discovered temperate Clostridioides difficile bacteriophage phiCDKH01 of the family Siphoviridae.

A temperate siphovirus, phiCDKH01, was obtained from a clinical isolate of Clostridioides difficile. The phage genome is a 45,089-bp linear double-stranded DNA molecule with an average G+C content of 28.7%. It shows low similarity to known phage genomes, except for phiCD24-1. Genomic and phylogenetic analysis revealed that phiCDKH01 is a newly discovered phage. Sixty-six putative ORFs were predicted in the genome, 37 of which code for proteins with predicted functions. The phiCDKH01 prophage was localized in the host genome. The results of this study increase our knowledge about the genetic diversity of tailed phages.

Examination of Staphylococcus aureus Prophages Circulating in Egypt.

Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton-Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt.

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing.

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

Comprehensive in silico survey of the Mycolicibacterium mobilome reveals an as yet underexplored diversity.

The mobilome plays a crucial role in bacterial adaptation and is therefore a starting point to understand and establish the gene flow occurring in the process of bacterial evolution. This is even more so if we consider that the mobilome of environmental bacteria can be the reservoir of genes that may later appear in the clinic. Recently, new genera have been proposed in the family Mycobacteriaceae, including the genus Mycolicibacterium, which encompasses dozens of species of agricultural, biotechnological, clinical and ecological importance, being ubiquitous in several environments. The current scenario in the Mycobacteriaceae mobilome has some bias because most of the characterized mycobacteriophages were isolated using a single host strain, and the few plasmids reported mainly relate to the genus Mycobacterium. To fill in the gaps in these issues, we performed a systematic in silico study of these mobile elements based on 242 available genomes of the genus Mycolicibacterium. The analyses identified 156 putative plasmids (19 conjugative, 45 mobilizable and 92 non-mobilizable) and 566 prophages in 86 and 229 genomes, respectively. Moreover, a contig was characterized by resembling an actinomycete integrative and conjugative element (AICE). Within this diversity of mobile genetic elements, there is a pool of genes associated with several canonical functions, in addition to adaptive traits, such as virulence and resistance to antibiotics and metals (mercury and arsenic). The type-VII secretion system was a common feature in the predicted plasmids, being associated with genes encoding virulent proteins (EsxA, EsxB, PE and PPE). In addition to the characterization of plasmids and prophages of the family Mycobacteriaceae, this study showed an abundance of these genetic elements in a dozen species of the genus Mycolicibacterium.

Characterization of φEf-vB1 prophage infecting oral Enterococcus faecalis and enhancing bacterial biofilm formation.

Introduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders1-4. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 109 per gram, and these numbers seem to persist throughout life5-7. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections8-10. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.

The enemy from within: a prophage of Roseburia intestinalis systematically turns lytic in the mouse gut, driving bacterial adaptation by CRISPR spacer acquisition.

Despite an overall temporal stability in time of the human gut microbiota at the phylum level, strong variations in species abundance have been observed. We are far from a clear understanding of what promotes or disrupts the stability of microbiome communities. Environmental factors, like food or antibiotic use, modify the gut microbiota composition, but their overall impacts remain relatively low. Phages, the viruses that infect bacteria, might constitute important factors explaining temporal variations in species abundance. Gut bacteria harbour numerous prophages, or dormant viruses, which can evolve to become ultravirulent phage mutants, potentially leading to important bacterial death. Whether such phenomenon occurs in the mammal's microbiota has been largely unexplored. Here we studied temperate phage-bacteria coevolution in gnotoxenic mice colonised with Roseburia intestinalis, a dominant symbiont of the human gut microbiota, and Escherichia coli, a sub-dominant member of the same microbiota. We show that R. intestinalis L1-82 harbours two active prophages, Jekyll and Shimadzu. We observed the systematic evolution in mice of ultravirulent Shimadzu phage mutants, which led to a collapse of R. intestinalis population. In a second step, phage infection drove the fast counter-evolution of host phage resistance mainly through phage-derived spacer acquisition in a clustered regularly interspaced short palindromic repeats array. Alternatively, phage resistance was conferred by a prophage originating from an ultravirulent phage with a restored ability to lysogenize. Our results demonstrate that prophages are a potential source of ultravirulent phages that can successfully infect most of the susceptible bacteria. This suggests that prophages can play important roles in the short-term temporal variations observed in the composition of the gut microbiota.

A Bioinformatic Analysis of Integrative Mobile Genetic Elements Highlights Their Role in Bacterial Adaptation.

Mobile genetic elements (MGEs) contribute to bacterial adaptation and evolution; however, high-throughput, unbiased MGE detection remains challenging. We describe MGEfinder, a bioinformatic toolbox that identifies integrative MGEs and their insertion sites by using short-read sequencing data. MGEfinder identifies the genomic site of each MGE insertion and infers the identity of the inserted sequence. We apply MGEfinder to 12,374 sequenced isolates of 9 prevalent bacterial pathogens, including Mycobacterium tuberculosis, Staphylococcus aureus, and Escherichia coli, and identify thousands of MGEs, including candidate insertion sequences, conjugative transposons, and prophage elements. The MGE repertoire and insertion rates vary across species, and integration sites often cluster near genes related to antibiotic resistance, virulence, and pathogenicity. MGE insertions likely contribute to antibiotic resistance in laboratory experiments and clinical isolates. Additionally, we identified thousands of mobility genes, a subset of which have unknown function opening avenues for exploration. Future application of MGEfinder to commensal bacteria will further illuminate bacterial adaptation and evolution.

R18C is a new viable P2-like bacteriophage of rabbit origin infecting Citrobacter rodentium and Shigella sonnei strains.

Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.

Large Phenotypic and Genetic Diversity of Prophages Induced from the Fish Pathogen Vibrio anguillarum.

Vibrio anguillarum is a marine pathogenic bacterium that causes vibriosis in fish and shellfish. Although prophage-like sequences have been predicted in V. anguillarum strains, many are not characterized, and it is not known if they retain the functional capacity to form infectious particles that can infect and lysogenize other bacterial hosts. In this study, the genome sequences of 28 V. anguillarum strains revealed 55 different prophage-related elements. Chemical and spontaneous induction allowed a collection of 42 phage isolates, which were classified in seven different groups according to a multiplex PCR assay. One shared prophage sequence, p41 (group III), was present in 17 V. anguillarum strains, suggesting that this specific element is very dynamically exchanged among V. anguillarum populations. Interestingly, the host range of genetically identical phages was highly dependent on the strains used for proliferation, indicating that phenotypic properties of phages were partly regulated by the host. Finally, experimental evidence displayed that the induced phage ɸVa_90-11-287_p41 was able to lysogenize V. anguillarum strain Ba35, and subsequently spontaneously become released from the lysogenized cells, demonstrating an efficient transfer of the phage among V. anguillarum strains. Altogether, the results showed large genetic and functional diversity and broad distribution of prophages in V. anguillarum, and demonstrated the potential of prophages as drivers of evolution in V. anguillarum strains.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

A prophage and two ICESa2603-family integrative and conjugative elements (ICEs) carrying optrA in Streptococcus suis.

OBJECTIVES: To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene optrA and identify the genetic elements involved in the horizontal transfer of the optrA gene in Streptococcus suis. METHODS: A total of 237 S. suis isolates were screened for the presence of the optrA gene by PCR. Whole-genome DNA of three optrA-positive strains was completely sequenced using the Illumina MiSeq and Pacbio RSII platforms. MICs were determined by broth microdilution. Transferability of the optrA gene in S. suis was investigated by conjugation. The presence of circular intermediates was examined by inverse PCR. RESULTS: The optrA gene was present in 11.8% (28/237) of the S. suis strains. In three strains, the optrA gene was flanked by two copies of IS1216 elements in the same orientation, located either on a prophage or on ICESa2603-family integrative and conjugative elements (ICEs), including one tandem ICE. In one isolate, the optrA-carrying ICE transferred with a frequency of 2.1 × 10-8. After the transfer, the transconjugant displayed elevated MICs of the respective antimicrobial agents. Inverse PCRs revealed that circular intermediates of different sizes were formed in the three optrA-carrying strains, containing one copy of the IS1216E element and the optrA gene alone or in combination with other resistance genes. CONCLUSIONS: A prophage and two ICESa2603-family ICEs (including one tandem ICE) associated with the optrA gene were identified in S. suis. The association of the optrA gene with the IS1216E elements and its location on either a prophage or ICEs will aid its horizontal transfer.

Characterization of Tn6349, a novel mosaic transposon carrying poxtA, cfr and other resistance determinants, inserted in the chromosome of an ST5-MRSA-II strain of clinical origin.

OBJECTIVES: To characterize the genetic element carrying the poxtA oxazolidinone resistance gene found in the poxtA index strain Staphylococcus aureus AOUC-0915 isolated from a cystic fibrosis patient. METHODS: The genetic context of poxtA was investigated by bioinformatics analysis of WGS data of strain AOUC-0915, followed by PCR and confirmatory Sanger sequencing for repetitive regions. Conjugation and electrotransformation experiments were carried out to assess horizontal transferability using S. aureus and Enterococcus faecalis recipients. Production of phage particles was evaluated by PCR using DNA preparations obtained after phage induction. Excision of the transposon carrying poxtA was evaluated by inverse PCR experiments for detection of circular intermediates. RESULTS: poxtA was found to be associated with a 48 kb composite transposon of original structure, named Tn6349, inserted into a φN315-like prophage. The transposon was bounded by two IS1216 insertion sequences, carried several resistance genes [erm(B), cfr, poxtA and fexB] and exhibited a mosaic structure made by a derivative of plasmid pE35048-oc (previously described in an Enterococcus faecium clinical isolate) and Tn6657, a novel composite transposon carrying the poxtA and fexB genes. Excision ability of Tn6349 as a circular intermediate was demonstrated. Transferability of Tn6349 or modules thereof to S. aureus or E. faecalis by either conjugation or electrotransformation was not detected. Induction of the φN315-like prophage carrying Tn6349 was not observed. CONCLUSIONS: This study describes the structure of Tn6349, a novel composite transposon carrying several resistance determinants to anti-ribosomal drugs, including cfr and poxtA, from an oxazolidinone-resistant MRSA strain. Analysis of Tn6349 revealed a modular structure that could favour the mobilization of its resistance determinants.

Diversity patterns of bacteriophages infecting Aggregatibacter and Haemophilus species across clades and niches.

Aggregatibacter and Haemophilus species are relevant human commensals and opportunistic pathogens. Consequently, their bacteriophages may have significant impact on human microbial ecology and pathologies. Our aim was to reveal the prevalence and diversity of bacteriophages infecting Aggregatibacter and Haemophilus species that colonize the human body. Genome mining with comparative genomics, screening of clinical isolates, and profiling of metagenomes allowed characterization of 346 phages grouped in 52 clusters and 18 superclusters. Less than 10% of the identified phage clusters were represented by previously characterized phages. Prophage diversity patterns varied significantly for different phage types, host clades, and environmental niches. A more diverse phage community lysogenizes Haemophilus influenzae and Haemophilus parainfluenzae strains than Aggregatibacter actinomycetemcomitans and "Haemophilus ducreyi". Co-infections occurred more often in "H. ducreyi". Phages from Aggregatibacter actinomycetemcomitans preferably lysogenized strains of specific serotype. Prophage patterns shared by subspecies clades of different bacterial species suggest similar ecoevolutionary drivers. Changes in frequencies of DNA uptake signal sequences and guanine-cytosine content reflect phage-host long-term coevolution. Aggregatibacter and Haemophilus phages were prevalent at multiple oral sites. Together, these findings should help exploring the ecoevolutionary forces shaping virus-host interactions in the human microbiome. Putative lytic phages, especially phiKZ-like, may provide new therapeutic options.

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.

A novel uncultured marine cyanophage lineage with lysogenic potential linked to a putative marine Synechococcus 'relic' prophage.

Marine cyanobacteria are important contributors to primary production in the ocean and their viruses (cyanophages) affect the ocean microbial communities. Despite reports of lysogeny in marine cyanobacteria, a genome sequence of such temperate cyanophages remains unknown although genomic analysis indicate potential for lysogeny in certain marine cyanophages. Using assemblies from Red Sea and Tara Oceans metagenomes, we recovered genomes of a novel uncultured marine cyanophage lineage, which contain, in addition to common cyanophage genes, a phycobilisome degradation protein NblA, an integrase and a split DNA polymerase. The DNA polymerase forms a monophyletic clade with a DNA polymerase from a genomic island in Synechococcus WH8016. The island contains a relic prophage that does not resemble any previously reported cyanophage but shares several genes with the newly identified cyanophages reported here. Metagenomic recruitment indicates that the novel cyanophages are widespread, albeit at low abundance. Here, we describe a novel potentially lysogenic cyanophage family, their abundance and distribution in the marine environment.

Characterization of Listeria prophages in lysogenic isolates from foods and food processing environments.

Prophages are commonly found in Listeria genomes, potentially enhancing survival or fitness of Listeria spp. Currently, there is still limited information on the distribution of prophages among Listeria isolates of different allelic types and from various sources. In this study, by using mitomycin C induction, prophages were found in 23/144 isolates (16.0%), including 13 L. monocytogenes and 10 Listeria spp. isolates, resulting in 28 and 11 induced phages, respectively. These prophage-carrying isolates (lysogens) were obtained from foods and food-related environments presenting 3 common allelic types (ATs) of L. monocytogenes (lineage I, II and IV), 4 ATs of L. innocua and 1 AT of L. welshimeri. The likelihood of prophage-carrying isolates of L. monocytogenes was 14.4 (95% CI: 4.9-35.4), and 18.5 (95% CI: 4.8-50.2) for Listeria spp. The 39 induced phages were classified into 3 lysis groups by the host range test against 9 major serotypes of L. monocytogenes and 5 species of Listeria. Most phages were host-specific with higher ability to lyse L. monocytogenes serotype 4 than other serotypes. The genome size of phages ranged from 35±2 kb to 50±2 kb and belonged to two common phage families, Myoviridae and Siphoviridae. Restriction analysis classified 19 selected phages into 16 restriction profiles, suggesting highly diverse prophages with at least 16 types. This may contribute to the variation in the genomes of Listeria. Information obtained here provides basic knowledge for further study to understand the overall role of prophages in Listeria, including roles in survival or fitness in foods and food processing environments.

Pelagiphages in the Podoviridae family integrate into host genomes.

The Pelagibacterales order (SAR11) in Alphaproteobacteria dominates marine surface bacterioplankton communities, where it plays a key role in carbon and nutrient cycling. SAR11 phages, known as pelagiphages, are among the most abundant phages in the ocean. Four pelagiphages that infect Pelagibacter HTCC1062 have been reported. Here, we report 11 new pelagiphages in the Podoviridae family. Comparative genomics classified these pelagiphages into the HTVC019Pvirus genus, which includes the previously reported pelagiphages HTVC011P and HTVC019P. Phylogenomic analysis clustered HTVC019Pvirus pelagiphages into three subgroups. Integrases were identified in all but one HTVC019Pvirus genome. Site-specific integration of HTVC019Pvirus pelagiphages into host tRNA genes was verified experimentally, demonstrating the capacity of these pelagiphages to propagate by both lytic and lysogenic infection. Evidence of pelagiphage integration was also retrieved from the Global Ocean Survey database, showing that prophages are found in natural SAR11 populations. HTVC019Pvirus pelagiphages could impact SAR11 populations by a variety of mechanisms, including mortality, genetic transduction and prophage-induced viral immunity. HTVC019Pvirus pelagiphages are a rare example of cultured lysogenic phage that can be implicated in ecological processes on broad scales. These pelagiphages have the potential to become a useful model for investigating strategies of host infection and phage-dependent horizontal gene transfer.