The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions.

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism.

Essential Steps in Characterizing Bacteriophages: Biology, Taxonomy, and Genome Analysis.

Because of the rise in antimicrobial resistance there has been a significant increase in interest in phages for therapeutic use. Furthermore, the cost of sequencing phage genomes has decreased to the point where it is being used as a teaching tool for genomics. Unfortunately, the quality of the descriptions of the phage and its annotation frequently are substandard. The following chapter is designed to help people working on phages, particularly those new to the field, to accurately describe their newly isolated viruses.

Comparative Analysis of 37 Acinetobacter Bacteriophages.

Members of the genus Acinetobacter are ubiquitous in the environment and the multiple-drug resistant species A. baumannii is of significant clinical concern. This clinical relevance is currently driving research on bacterial viruses infecting A. baumannii, in an effort to implement phage therapy and phage-derived antimicrobials. Initially, a total of 42 Acinetobacter phage genome sequences were available in the international nucleotide sequence databases, corresponding to a total of 2.87 Mbp of sequence information and representing all three families of the order Caudovirales and a single member of the Leviviridae. A comparative bioinformatics analysis of 37 Acinetobacter phages revealed that they form six discrete clusters and two singletons based on genomic organisation and nucleotide sequence identity. The assignment of these phages to clusters was further supported by proteomic relationships established using OrthoMCL. The 4067 proteins encoded by the 37 phage genomes formed 737 groups and 974 orphans. Notably, over half of the proteins encoded by the Acinetobacter phages are of unknown function. The comparative analysis and clustering presented enables an updated taxonomic framing of these clades.

Taxonomic reassessment of N4-like viruses using comparative genomics and proteomics suggests a new subfamily - "Enquartavirinae".

The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: "G7cvirus" (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), "Lit1virus" (LIT1, PA26 and vB_PaeP_C2-10_Ab09), "Sp58virus" (SP058 and SP076), and "Dss3virus" (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of "G7cvirus", Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the "Enquartavirinae".

A proposed new bacteriophage subfamily: "Jerseyvirinae".

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB_SenS_AG11, vB_SenS-Ent1, vB_SenS-Ent2, vB_SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus "Jerseylikevirus". The proposed genus "K1glikevirus" consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus "Sp3unalikevirus" contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6 kb (49.6-51.4 mol % G+C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic.

Characterization of two temperate Lactobacillus paracasei bacteriophages: morphology, kinetics and adsorption.

BACKGROUND/AIMS: Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate Lactobacillus paracasei phages Φ iLp84 and Φ iLp1308, infective for probiotic strains. METHODS: Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. RESULTS: Phages Φ iLp84 and Φ iLp1308 belong to the Siphoviridae family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ iLp84, and 51 min, 92 min and 28 for Φ iLp1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ iLp84 produced plaques on only 1 out of 8 strains tested. CONCLUSION: Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate L. paracasei phages of similar origin.

Integration of genomic and proteomic analyses in the classification of the Siphoviridae family.

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.

Characterization of Staphylococcus epidermidis phage vB_SepS_SEP9 - a unique member of the Siphoviridae family.

Relatively few phages (<10) of coagulase negative staphylococci (CoNS) have been described. Staphylococcus epidermidis phage vB_SepS_SEP9 is a siphovirus with a unique morphology as a staphylococcal phage, possessing a very long tail. Its genome is unique and unrelated to any phage genomes deposited in public databases. It appears to encode a nonfunctional integrase. Due to the not having a recognizable lysogeny module, the phage is unable lysogenize. The genome comprises 129 coding sequences (CDS), 46 of which have an assigned function and 59 are unique. Its unique morphology and genome led to the proposal of the establishment of a new Siphoviridae genus named "Sep9likevirus".

Supersize me: Cronobacter sakazakii phage GAP32.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.

Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus "Twort-like viruses," three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

The genome and proteome of Serratia bacteriophage η which forms unstable lysogens.

BACKGROUND: Serratia marcescens phage η is a temperate unclassified member of the Siphoviridae which had been reported as containing hypermodified guanine residues. METHODS: The DNA was characterized by enzymatic digestion followed by HPLC analysis of the nucleoside composition, and by DNA sequencing and proteomic analysis. Its ability to form stable lysogens and integrate was also investigated. RESULTS: Enzymatic digestion and HPLC analysis revealed phage η DNA did not contain modified bases. The genome sequence of this virus, determined using pyrosequencing, is 42,724 nucleotides in length with a mol% GC of 49.9 and is circularly permuted. Sixty-nine putative CDSs were identified of which 19 encode novel proteins. While seven close genetic relatives were identified, they shared sequence similarity with only genes 40 to 69 of the phage η genome, while gp1 to gp39 shared no conserved relationship. The structural proteome, determined by SDS-PAGE and mass spectrometry, revealed seven unique proteins. This phage forms very unstable lysogens with its host S. marcescens.

Isolation and characterization of a new Staphylococcus epidermidis broad-spectrum bacteriophage.

Staphylococcus epidermidis is considered an important nosocomial pathogen, being very tolerant to the host immune system and antibiotherapy, particularly when in biofilms. Due to its high resistance, alternative antimicrobial strategies are under development. The use of bacteriophages is seen as an important strategy to combat pathogenic organisms. In this study, a S. epidermidis myovirus, SEP1, was isolated and characterized. The genome of this phage was sequenced and shown to be related peripherally to the genus Twortlikevirus. However, when compared with other phages of this genus, it showed DNA sequence identities no greater than 58.2 %. As opposed to other polyvalent viruses of the genus Twortlikevirus, SEP1 is highly specific to S. epidermidis strains. The good infectivity shown by this phage as well as its high lytic spectrum suggested that it might be a good candidate for therapeutic studies.

A suggested classification for two groups of Campylobacter myoviruses.

Most Campylobacter bacteriophages isolated to date have long contractile tails and belong to the family Myoviridae. Based on their morphology, genome size and endonuclease restriction profile, Campylobacter phages were originally divided into three groups. The recent genome sequencing of seven virulent campylophages reveal further details of the relationships between these phages at the genome organization level. This article details the morphological and genomic features among the campylophages, investigates their taxonomic position, and proposes the creation of two new genera, the "Cp220likevirus" and "Cp8unalikevirus" within a proposed subfamily, the "Eucampyvirinae"

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7.

Bacteriophages are considered as promising biological agents for the control of infectious diseases. Sequencing of their genomes can ascertain the absence of antibiotic resistance, toxin or virulence genes. The anti-O157 : H7 coliphage, PhaxI, was isolated from a sewage sample in Iran. Morphological studies by transmission electron microscopy showed that it has an icosahedral capsid of 85-86 nm and a contractile tail of 115×15 nm. PhaxI contains dsDNA composed of 156 628 nt with a G+C content of 44.5 mol% that encodes 209 putative proteins. In MS analysis of phage particles, 92 structural proteins were identified. PhaxI lyses Escherichia coli O157 : H7 in Luria-Bertani medium and milk, has an eclipse period of 20 min and a latent period of 40 min, and has a burst size of about 420 particles per cell. PhaxI is a member of the genus 'Viunalikevirus' of the family Myoviridae and is specific for E. coli O157 : H7.

The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5.

BACKGROUND: Bacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs. METHODS: We examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set. RESULTS: Phage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.

The Genome of Cronobacter sakazakii Bacteriophage vB_CsaP_GAP227 Suggests a New Genus within the Autographivirinae.

The genome of Cronobacter sakazakii podovirus vB_CsaP_GAP227 was fully sequenced. The DNA of this lytic phage consists of 41,796 bp and has a G+C content of 55.7%. Forty-nine open reading frames and no tRNAs were identified. This phage is related to Yersinia phages ϕR8-01 and ϕ80-18 and Aeromonas phage phiAS7.

Virus species polemics: 14 senior virologists oppose a proposed change to the ICTV definition of virus species.

The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) has recently decided to modify the current definition of virus species (Code of Virus Classification and Nomenclature Rule 3.21) and will soon ask the full ICTV membership (189 voting members) to ratify the proposed controversial change. In this discussion paper, 14 senior virologists, including six Life members of the ICTV, compare the present and proposed new definition and recommend that the existing definition of virus species should be retained. Since the pros and cons of the proposal posted on the ICTV website are not widely consulted, the arguments are summarized here in order to reach a wider audience.

Complete genome sequence of Cronobacter sakazakii bacteriophage vB_CsaM_GAP161.

Cronobacter sakazakii is an opportunistic pathogen that causes infant meningitis and is often associated with milk-based infant formula. We have fully sequenced the genome of a newly isolated lytic C. sakazakii myovirus, vB_CsaM_GAP161, briefly named GAP161. It consists of 178,193 bp and has a G+C content of 44.5%. A total of 277 genes, including 275 open reading frames and two tRNA-encoding genes, were identified. This phage is closely related to coliphages RB16 and RB43 and Klebsiella pneumoniae phage KP15.