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Vibrio phage KIT04 was isolated from muscle tissue samples collected from a local market in Vietnam. KIT04 is a lytic phage that is specific to Vibrio parahaemolyticus. The one-step growth curve determined the burst size and latent period of 0.01 multiplicity of infection KIT04 in V. parahaemolyticus as approximately 156 plaque-forming units/bacterium and 45 min, respectively. Vibrio phage KIT04 has an approximately 76.4 ± 4.5 nm diameter icosahedral head and a tail length of approximately 159.5 ± 16.6 nm long tail. KIT04 significantly reduced V. parahaemolyticus ATCC 17802 in vitro. Complete genome analysis showed that KIT04 had a 114,933 bp dsDNA genome with 40.24% G + C content and 160 open reading frames (ORFs). However, the phage genome contained 24 tRNAs and no lysogeny-related genes. Moreover, five of the 160 ORFs encoded unique hypothetical proteins, indicating that KIT04 is a novel phage. Genomic comparison indicated that KIT04 is closely related to the Vibrio phages pVp-1 and VPT02. Further, phylogenetic analysis of the major tail proteins and whole genome supported the KIT04 classification into the subfamily Ermolyevavirinae. Our study describes a new candidate phage that could be used as a bioagent for controlling Vibrio pathogens. • Vibrio phage KIT04 was isolated using a Vibrio parahaemolyticus strain as a host. • Vibrio phage KIT04 is specific to V. parahaemolyticus. • Vibrio phage KIT04 is a novel phage of the subfamily Ermolyevavirinae. [ABSTRACT FROM AUTHOR]

Methicillin resistance Staphylococcus aureus (MRSA) is currently threatening global public health, and a similar issue was encountered in Hainan. For establishing a promising alternative therapeutic option, a phage with the capacity of lysing 18 out of 35 MRSA clinical isolates was recovered from domestic natural sources, which was termed as vB_SauP_L1 due to its morphology and genomic similarity with Rountreeviridae. Satisfactory proliferation rate and environmental tolerance were demonstrated by subsequent infectious properties tests and stability assessments. Sequencing revealed that its genome consisted of a linear double-stranded DNA of 17,114 bp, in which neither virulent nor resistant gene was detected, indicating its potential in MRSA prevention and treatment. • vB_SauP_L1 is the first lytic phage against methicillin-resistant Staphylococcus aureus in Hainan Island. • It is characterised by a lysis rate of 51.4% (n = 35), broad pH and temperature stabilities. • As a Rakietenvirinae member of the family Rountreeviridae , its genome consists of a linear double-stranded DNA of 17,114 bp. [ABSTRACT FROM AUTHOR]

The increasing prevalence of drug-resistant Escherichia coli (E. coli) resulting from the excessive utilization of antibiotics necessitates the immediate exploration of alternative approaches to counteract pathogenic E. coli. Phages, with their unique antibacterial mechanisms, are considered promising candidates for treating bacterial infections. Herein, we isolated a lytic Escherichia phage Tequatrovirus YZ2 (phage YZ2), which belongs to the genus Tequatrovirus. The genome of phage YZ2 consists of 168,356 base pairs with a G + C content of 35.34% and 269 putative open reading frames (ORFs). Of these, 146 ORFs have been annotated as functional proteins associated with nucleotide metabolism, structure, transcription, DNA replication, translation, and lysis. In the mouse model of a skin wound infected by E. coli , phage YZ2 therapy significantly promoted the wound healing. Furthermore, histopathological analysis revealed reductions in IL-1β and TNF-α and increased VEGF levels, indicating the potential of phages as effective antimicrobial agents against E. coli infection. • Escherichia coli is a common pathogen that causes skin wound infections, posing a threat to human health. • Tequatrovirus YZ2 exhibits statistically significant antibacterial activity against Escherichia coli (ATCC25922). • In vivo experiments demonstrated the potential of Tequatrovirus YZ2 for treating Escherichia coli skin wound infections. [ABSTRACT FROM AUTHOR]

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4–60 °C) and broad pH stability (4.0–12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3–6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens. • vB_SalS_PSa2 is a newly isolated Demerecviridae phage belonging to the T5 group. • vB_SalS_PSa2 displays a wide host range to tested clinical isolates of Salmonella and E. coli , most being multidrug resistant. • vB_SalS_PSa2 is stable in a relative broad temperature and pH range. • vB_SalS_PSa shows good antibacterial effect in milk under refrigerated conditions. • vB_SalS_PSa2 shows good inhibition effect on biofilm formation and degradation on the mature biofilms of the tested targets. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the most important pathogens of healthcare-associated infections. The rising prevalence of multidrug-resistant A. baumannii (MRAB) strains and biofilm formation impact the outcome of conventional treatment. Phage-related therapy is a promising strategy to tame troublesome multidrug-resistant bacteria. Here, we isolated and evaluated a highly efficient lytic phage called MRABP9 from hospital sewage. The phage was a novel species within the genus Friunavirus and exhibited lytic activity against 2 other identified MRAB strains. Genomic analysis revealed it was a safe virulent phage and a pectate lyase domain was identified within its tail spike protein. MRABP9 showed potent bactericidal and anti-biofilm activity against MRAB, significantly delaying the time point of bacterial regrowth in vitro. Phage administration could rescue the mice from acute lethal MRAB infection. Considering its features, MRABP9 has the potential as an efficient candidate for prophylactic and therapeutic use against acute infections caused by MRAB strains. • The MRAB phage MRABP9 is a novel species within genus Friunavirus. • MRABP9 delayed the emergence of drug-resistant bacterial strain. • MRABP9 showed potent bactericidal activity against MRAB. • Genomic analysis of MRABP9 revealed its safety and lytic feature. • MRABP9 significantly inhibited and destroyed bacterial biofilm. [ABSTRACT FROM AUTHOR]

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%–1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications. • Huma & Simurgh had genome sizes of 16,853 and 17,245 bp and GC content of 29%. • No lysogeny-related, nor virulence genes were identified in their genomes. • They had latent period of 80 and 60 min and burst sizes of 45 and 40 PFU/ml. [ABSTRACT FROM AUTHOR]

The hydrophobicity of virions is a major physicochemical parameter regulating their dissemination in humans and the environment. But knowledge about potential factors modulating virion hydrophobicity is limited due to the lack of suitable quantifying methods. It has been recently shown that sodium dodecyl-sulfate (SDS) labels capsid hydrophobic domains in capillary zone electrophoresis of non-enveloped virions, altering their electrophoretic mobility (μ) in proportion to their hydrophobicity. This was exploited here to quantify the hydrophobicity of GA, Qβ and MS2 phages as a function of pH. By subtracting the native from the SDS-modified μ of phages, measured in the absence and presence of SDS, respectively, we defined a "hydrophobic index" increasing with virion hydrophobicity. Using this approach, we found that the virion hydrophobicity changes at a virion-specific pivotal pH. This procedure may be applied under various physicochemical conditions and to diverse non-enveloped virus families of significance to human health and the environment. [Display omitted] • A new method to measure hydrophobicity of non-enveloped virions is provided. • This method is adaptable to various physicochemical conditions in aqueous media. • pH-dependent features in hydrophobicity of MS2, Qβ and GA phages are reported. • Virion's hydrophobicity may change especially at specific pH called "pivotal". • Virions ionic status and hydrophobicity change differently with pH. [ABSTRACT FROM AUTHOR]

CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the "HK97-fold" shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain ("I-domain"), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently.

The multifunctional phage replication protein gp43 is composed of an N-terminal prim-pol domain and a C-terminal domain similar to the SF4-type replicative helicases. We prepared four mutants all missing the prim-pol domain with the helicase core flanked by accessory N- and C-terminal regions truncated to varying extents. The shortest fragment still possessing strong ssDNA-dependent ATPase activity and helicase activity was gp43HEL519-983. The other proteins tested were gp43HEL557-983, gp43HEL519-855 and gp43HEL519-896. Removal of the 38 N-terminal residues in gp43HEL557-983, or the 128 and 87 C-terminal residues in gp43HEL519-855 and gp43HEL519-896, resulted in a significant decrease in the ATPase activities. The 38-amino acid N-terminal region has probably a function in modulating DNA binding and protein oligomerization. Deletion of the 87 C-terminal residues resulted in a twofold increase in the unwinding rate. This region is likely indispensable for binding to DNA substrates. [Display omitted] Four deletion mutants of a phage SF4 family helicase were characterized All mutants contain a helicase core and truncated accessory N- or C-terminal regions The mutant activities, oligomerization and DNA binding properties differ markedly The N- and C-terminal sequences are likely involved in fine tuning protein properties [ABSTRACT FROM AUTHOR]

Erwinia and Pantoea are closely related bacterial plant pathogens in the Gram negative Enterobacteriales order. Sixty tailed bacteriophages capable of infecting these pathogens have been completely sequenced by investigators around the world and are in the current databases, 30 of which were sequenced by our lab. These 60 were compared to 991 other Enterobacteriales bacteriophage genomes and found to be, on average, just over twice the overall average length. These Erwinia and Pantoea phages comprise 20 clusters based on nucleotide and protein sequences. Five clusters contain only phages that infect the Erwinia and Pantoea genera, the other 15 clusters are closely related to bacteriophages that infect other Enterobacteriales ; however, within these clusters the Erwinia and Pantoea phages tend to be distinct, suggesting ecological niche may play a diversification role. The failure of many of their encoded proteins to have predicted functions highlights the need for further study of these phages. [ABSTRACT FROM AUTHOR]

The ΦX174 reduction effect describes a plasmid-based inhibitory phenomenon that mimics the superinfection inhibition found in wild phage populations. In this effect, when a portion of the ΦX174 genome – the 3′ end of the pilot protein gene (H), the 5' end of the replication gene (A), and the H-A intergenic region – is present on a plasmid in the host cell, almost complete protection from phage infection occurs. Here we demonstrate that only the phage pilot protein H portion of the plasmid is sufficient for the observed inhibition, that protein synthesis is necessary for inhibition to occur, that inserting the entire H gene in the plasmid may also impart a blocking effect, and that partial to complete recovery from this inhibition is possible with minimal viral evolution. [ABSTRACT FROM AUTHOR]

Abstract Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of N = 12 complete chicken PBVs (ChPBV) segments (N = 5 of S1 and N = 7 of S2, Acc. Nos.: MH425579-90) were determined using viral metagenomic and RT-PCR techniques from a single cloacal sample of a diarrheic chicken. The identified ChPBV segments are unrelated to each other and distant from all of the currently known PBVs. In silico sequence analyses revealed the presence of conserved prokaryotic Shine-Dalgarno-like (SD-like) sequences upstream of the three presumed open reading frames (ORFs) of the S1 and a single presumed ORF of the S2 segments. According to the results of expression analyses in E. coli using 6xHis-tagged recombinant ChPBV segment 1 construct and Western blot these SD-like sequences are functional in vivo suggesting that S1 of study PBVs can contain three ORFs and supporting the bacteriophage-nature of PBVs. Highlights • Twelve novel picobirnavirus segments were identified from a chicken cloacal sample. • Full-length sequences of five segment 1 and seven segment 2 were determined. • Prokaryotic Shine-Dalgarno-like (SD-like) sequences were present upstream of all ORFs. • In vivo expression analysis indicates the SD-like sequences are functional in E. coli. • The presence of functional SD-like sequences further support the phage nature of PBVs. [ABSTRACT FROM AUTHOR]

Tectiviridae are composed of tailless bacteriophages with an icosahedral capsid and an inner membrane enclosing a double-stranded 15 kb linear DNA genome. Five of the seven previously studied Tectivirus isolates infect bacteria from Bacillus cereus sensu lato group (Betatectivirus), one distantly related member (PRD1) infect Enterobactericeae (Alpatectivirus) and one recently discovered virus infect Gluconobacter cerinus (Gammatectivirus). Here we expand the host spectrum of Betatectivirus elements to four additional genera ( Streptococcus , Exiguobacterium , Clostridium and Brevibacillus ) and to more distantly related Bacillus species ( B. pumilus and B. flexus ) by studying the genomes of fourteen novel tectiviral elements. Overall, the genomes show significant conservation in gene synteny and in modules responsible for genome replication and formation of the virion core (including DNA packaging). Notable variation exists in regions encoding host attachment and lysis along with the surrounding area of a site in which mutations are known to alter phage life cycle. [ABSTRACT FROM AUTHOR]

Currently, the Leviviridae and Cystoviridae are the only two recognized families of prokaryotic RNA viruses. Picobirnaviruses, which are bisegmented double-stranded RNA viruses commonly found in animal stool samples, are currently thought to be animal viruses, but have not been propagated in cell culture or in an animal model. We hypothesize that picobirnaviruses are prokaryotic RNA viruses. We identified and analyzed the genomes of 38 novel picobirnaviruses and determined that a classical bacterial sequence motif, the ribosomal binding site (RBS), is present in the 5′ untranslated regions (5′ UTRs) of all of the novel as well as all previously published picobirnavirus sequences. Among all viruses, enrichment of the RBS motif is only observed in viral families that infect prokaryotes and not in eukaryotic infecting viral families. These results will enable future studies to more accurately understand the biology of picobirnaviruses. [ABSTRACT FROM AUTHOR]

The temperate Escherichia coli bacteriophage D6 can exist as a circular plasmid prophage, and we report here its 91,159 bp complete genome sequence. It is a distant relative of the well-studied phage P1, but it is sufficiently different that it typifies a previously undescribed tailed phage type or cluster. Examination of the database of bacterial genome sequences revealed that phage P1 and D6 prophage plasmids are common in the Enterobacteriales, and in addition, previously described Salmonella phage SSU5 represents a different type of temperate tailed phage with a circular plasmid prophage that is also very common in this host order. This analysis also discovered additional divergent clusters of putative circular plasmid prophages within the two larger P1 and SSU5 groups (superclusters) that inhabit the Enterobacteriales as well as bacteria in several other orders in the Gamma-proteobacteria class. Very few of these sequences are annotated as putative prophages. [ABSTRACT FROM AUTHOR]

Myoviridae bacteriophages have a special contractile tail machine that facilitates high viral infection efficiency. The major component of this machine is a tail sheath that contracts during infection, allowing delivery of viral DNA into the host cell. Tail sheaths of Myoviridae phages are composed of multiple copies of individual proteins. The giant Pseudomonas aeruginosa phage PaBG is notable in its possession of two tail sheath proteins. These tail sheath proteins are encoded by orf 76 and 204 , which were cloned and expressed individually and together in Escherichia coli . We demonstrate that only co-expression of both genes results in efficient assembly of thermostable and proteolytically resistant polysheaths composed of gp76 and gp204 with approximately 1:1 stoichiometry. Both gp76 and gp204 have been identified as structural components of the virion particle. We conclude that during PaBG morphogenesis in vivo two proteins, gp76 and gp204, assemble the tail sheath. [ABSTRACT FROM AUTHOR]

Double-stranded DNA bacteriophages employ holin and endolysin functions to lyse host bacteria after virus multiplication. Holins oligomerize in the cytoplasmic membrane and trigger to form holes that cause cell death. For most systems these holes are also required for endolysin release to the cell wall, where it cleaves the peptidoglycan network. Orfs 25 and 26 of Bacillus subtilis phage SPP1 were predicted to encode the endolysin and holin functions, respectively. However, the product of the upstream orf 24.1 exhibits also holin features. We show that production of gp24.1 or gp26 in B. subtilis causes no major impact on cell growth, despite their ability to insert in the cytoplasmic membrane. Instant growth cessation and cell death is observed only upon coproduction of the two holin-like proteins. Surprisingly, a constitutive promoter was identified within orf 24.1, which we propose to correspond to the previously described SPP1 early promoter PE5. [ABSTRACT FROM AUTHOR]

We identified 9371 tailed phage prophages of 20 known types in reported complete genome sequences of 3298 bacteria in the Salmonella genus. These include 4758 P2 type and 744 P22 type prophages. The latter prophage types were found in the genome sequences of 127 and 24 bacterial host genera, increasing the known host ranges of phages in these groups by 114 and 20 genera, respectively. These prophage nucleotide sequences displayed much more diversity than was previously known from the 48 P2 and 24 P22 type authentic phages whose genomes have been sequenced. More detailed analysis of these prophage sequences indicated that major capsid protein (MCP) gene exchange between tailed phage clusters or types is extremely rare and that P22 prophage-encoded tailspikes correspond perfectly with their hosts’ surface polysaccharide structure; thus, MCP and tailspike sequences accurately predict tailed phage type (and thus lifestyle) and host cell surface polysaccharide structure, respectively. [ABSTRACT FROM AUTHOR]

Bacteriophage AR9 and its close relative PBS1 have been extensively used to construct early Bacillus subtilis genetic maps. Here, we present the 251,042 bp AR9 genome, a linear, terminally redundant double-stranded DNA containing deoxyuridine instead of thymine. Multiple AR9 genes are interrupted by non-coding sequences or sequences encoding putative endonucleases. We show that these sequences are group I and group II self-splicing introns. Eight AR9 proteins are homologous to fragments of bacterial RNA polymerase (RNAP) subunits β/β’. These proteins comprise two sets of paralogs of RNAP largest subunits, with each paralog encoded by two disjoint phage genes. Thus, AR9 is a phiKZ-related giant phage that relies on two multisubunit viral RNAPs to transcribe its genome independently of host transcription apparatus. Purification of one of PBS1/AR9 RNAPs has been reported previously, which makes AR9 a promising object for further studies of RNAP evolution, assembly and mechanism. [ABSTRACT FROM AUTHOR]

Bacteriophages use most frequently a tail apparatus to create a channel across the entire bacterial cell envelope to transfer the viral genome to the host cell cytoplasm, initiating infection. Characterization of this critical step remains a major challenge due to the difficulty to monitor DNA entry in the bacterium and its requirements. In this work we developed a new method to study phage DNA entry that has the potential to be extended to many tailed phages. Its application to study genome delivery of bacteriophage SPP1 into Bacillus subtilis disclosed a key role of the host cell membrane potential in the DNA entry process. An energized B. subtilis membrane and a millimolar concentration of calcium ions are shown to be major requirements for SPP1 DNA entry following the irreversible binding of phage particles to the receptor YueB. [ABSTRACT FROM AUTHOR]

The Microviridae , comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries. [ABSTRACT FROM AUTHOR]

The virion proteins of Pseudoalteromonas phage φRIO-1 were identified and quantitated by mass spectrometry and gel densitometry. Bioinformatic methods customized to deal with extreme divergence defined a φRIO-1 tail structure homology group of phages, which was further related to T7 tail and internal virion proteins (IVPs). Similarly, homologs of tubular tail components and internal virion proteins were identified in essentially all completely sequenced podoviruses other than those in the subfamily Picovirinae. The podoviruses were subdivided into several tail structure homology groups, in addition to the RIO-1 and T7 groups. Molecular phylogeny indicated that these groups all arose about the same ancient time as the φRIO-1/T7 split. Hence, the T7-like infection mechanism involving the IVPs was an ancestral property of most podoviruses. The IVPs were found to variably host both tail lysozyme domains and domains destined for the cytoplasm, including the N4 virion RNA polymerase embedded within an IVP-D homolog. [ABSTRACT FROM AUTHOR]

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. [ABSTRACT FROM AUTHOR]

Phage lambda׳s cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos ( cos N15 ) is closely related to cos λ , but whereas the cosB N15 subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB λ . A bioinformatic study of N15-like phages indicates that cosB N15 also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. [ABSTRACT FROM AUTHOR]

Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda׳s life cycle. [ABSTRACT FROM AUTHOR]

For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. [ABSTRACT FROM AUTHOR]

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae . These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. [ABSTRACT FROM AUTHOR]

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: ΦM12 is the first example of a T=19l geometry capsid, encapsulating the recently sequenced genome. Here, we present structures determined by cryo-EM of full and empty capsids. The structure reveals the pattern for assembly of 1140 HK97-like capsid proteins, pointing to interactions at the pseudo 3-fold symmetry axes that hold together the asymmetric unit. The particular smooth surface of the capsid, along with a lack of accessory coat proteins encoded by the genome, suggest that this interface is the primary mechanism for capsid assembly. Two-dimensional averages of the tail, including the neck and baseplate, reveal that ΦM12 has a relatively narrow neck that attaches the tail to the capsid, as well as a three-layer baseplate. When free from DNA, the icosahedral edges expand by about 5nm, while the vertices stay at the same position, forming a similarly smooth, but bowed, T=19l icosahedral capsid. [Copyright &y& Elsevier]

Abstract: Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12’s novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria. [Copyright &y& Elsevier]

Abstract: Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981. [Copyright &y& Elsevier]

Abstract: Bacteriophage particles contain both simple and complex macromolecular assemblages and machines that enable them to regulate the infection process under diverse environmental conditions with a broad range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible to observe the interactions of bacteriophages with their host cells under native-state conditions at unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of investigation and future directions in the field are also discussed. [Copyright &y& Elsevier]

Abstract: We report the study of phage AF, the first member of the canonical lambdoid phage group infecting Pseudomonas putida. Its 42.6kb genome is related to the “epsilon15-like viruses” and the “BPP-1-like viruses”, a clade of bacteriophages shaped by extensive horizontal gene transfer. The AF virions display exopolysaccharide (EPS)-degrading activity, which originates from the action of the C-terminal domain of the tail spike (Gp19). This protein shows high similarity to the tail spike of the T7-like P. putida-infecting phage φ15. These unrelated phages have an identical host spectrum and EPS degradation characteristics, designating the C-terminal part of Gp19 as sole determinant for these functions. While intact AF particles have biofilm-degrading properties, Gp19 and non-infectious AF particles do not, emphasizing the role of phage amplification in biofilm degradation. [Copyright &y& Elsevier]

Abstract: The complete genome sequences of over 220 mycobacteriophages reveal them to be highly diverse, with numerous types sharing little or no nucleotide sequence identity with each other. We have determined the preferences of these phages for Mycobacterium tuberculosis and for other strains of Mycobacterium smegmatis, and find there is a correlation between genome type (cluster, subcluster, singleton) and host range. For many of the phages, expansion of host range occurs at relatively high frequencies, and we describe several examples in which host constraints occur at early stages of infection (adsorption or DNA injection), and phages have the ability to expand their host range through mutations in tail genes. We present a model in which phage diversity is a function of both the ability of phages to rapidly adapt to new hosts and the richness of the diversity of the bacterial population from which those phages are isolated. [Copyright &y& Elsevier]

Abstract: In vitro assembly of bacteriophage P22 procapsids requires coat protein and sub-stoichiometric concentrations of the internal scaffolding protein. If there is no scaffolding protein, coat protein assembles aberrantly, but only at higher concentrations. Too much scaffolding protein results in partial procapsids. By treating the procapsid as a lattice that can bind and be stabilized by scaffolding protein we dissect procapsid assembly as a function of protein concentration and scaffolding/coat protein ratio. We observe that (i) the coat–coat association is weaker for procapsids than for aberrant polymer formation, (ii) scaffolding protein makes a small but sufficient contribution to stability to favor the procapsid form, and (iii) there are multiple classes of scaffolding protein binding sites. This approach should be applicable to other heterogeneous virus assembly reactions and will facilitate our ability to manipulate such in vitro reactions to probe assembly, and for development of nanoparticles. [Copyright &y& Elsevier]

Abstract: The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T=16 icosahedron attached to a contractile tail via a head–tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid. [Copyright &y& Elsevier]

Abstract: One of the outstanding questions in biology today is the origin of viruses. We have discovered a protein in the hyperthermophile Sulfolobus solfataricus while following proteome regulation during viral infection that led to the discovery of a fossil provirus. Characterization of the wild type and recombinant protein revealed that it assembled into virus-like particles with a diameter of ~32nm. Sequence and structural analyses showed that the likely proviral capsid protein, Sso2749, is homologous to a protein from Pyrococcus furiosus that forms virus-like particles using the HK-97 major capsid protein fold. The SsP2-provirus appears mosaic and contains proteins with similarity to, among others, eukaryotic herpesviruses and tailed dsDNA bacteriophage families, reinforcing the hypothesis of a common ancestral gene pool across all three domains of life. This is the first description of the HK-97 fold in a crenarchaeal virus and the first direct genomic connection of linocin-like protein cages to a virus. [Copyright &y& Elsevier]

Abstract: The mosaic composition of the genomes of dsDNA tailed bacteriophages (Caudovirales) is well known. Observations of this mosaicism have generally come from comparisons of small numbers of often rather distantly related phages, and little is known about the frequency or detailed nature of the processes that generate this kind of diversity. Here we review and examine the mosaicism within fifty-seven clusters of virion assembly genes from bacteriophage P22 and its “close” relatives. We compare these orthologous gene clusters, discuss their surprising diversity and document horizontal exchange of genetic information between subgroups of the P22-like phages as well as between these phages and other phage types. We also point out apparent restrictions in the locations of mosaic sequence boundaries in this gene cluster. The relatively large sample size and the fact that phage P22 virion structure and assembly are exceptionally well understood make the conclusions especially informative and convincing. [Copyright &y& Elsevier]

Abstract: Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven''t been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379–394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state. [Copyright &y& Elsevier]

Abstract: Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome. [ABSTRACT FROM AUTHOR]

Abstract: We present a detailed analysis of the genome architecture, structural proteome and infection-related properties of three Pseudomonas phages, designated LUZ7, LIT1 and PEV2. These podoviruses encapsulate 72.5 to 74.9kb genomes and lyse their host after 25min aerobic infection. PEV2 can successfully infect under anaerobic conditions, but its latent period is tripled, the lysis proceeds far slower and the burst size decreases significantly. While the overall genome structure of these phages resembles the well-studied coliphage N4, these Pseudomonas phages encode a cluster of tail genes which displays significant similarity to a Pseudomonas aeruginosa (cryptic) prophage region. Using ESI-MS/MS, these tail proteins were shown to be part of the phage particle, as well as ten other proteins including a giant 370kDa virion RNA polymerase. These phages are the first described representatives of a novel kind of obligatory lytic P. aeruginosa-infecting phages, belonging to the widespread “N4-like viruses” genus. [ABSTRACT FROM AUTHOR]