In Situ Structures of the Ultra-Long Extended and Contracted Tail of Myoviridae Phage P1.

The Myoviridae phage tail is a common component of contractile injection systems (CISs), essential for exerting contractile function and facilitating membrane penetration of the inner tail tube. The near-atomic resolution structures of the Myoviridae tail have been extensively studied, but the dynamic conformational changes before and after contraction and the associated molecular mechanism are still unclear. Here, we present the extended and contracted intact tail-structures of Myoviridae phage P1 by cryo-EM. The ultra-long tail of P1, 2450 Å in length, consists of a neck, a tail terminator, 53 repeated tail sheath rings, 53 repeated tube rings, and a baseplate. The sheath of the contracted tail shrinks by approximately 55%, resulting in the separation of the inner rigid tail tube from the sheath. The extended and contracted tails were further resolved by local reconstruction at 3.3 Å and 3.9 Å resolutions, respectively, allowing us to build the atomic models of the tail terminator protein gp24, the tube protein BplB, and the sheath protein gp22 for the extended tail, and of the sheath protein gp22 for the contracted tail. Our atomic models reveal the complex interaction network in the ultra-long Myoviridae tail and the novel conformational changes of the tail sheath between extended and contracted states. Our structures provide insights into the contraction and stabilization mechanisms of the Myoviridae tail.

Fine structure and assembly pattern of a minimal myophage Pam3.

The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 Å in diameter, connected via a three-section neck to an 840-Å-long contractile tail, ending with a three-module baseplate composed of only six protein components. This simplified baseplate consists of a central hub-spike surrounded by six wedge heterotriplexes, to which twelve tail fibers are covalently attached via disulfide bonds in alternating upward and downward configurations. In vitro reduction assays revealed a putative redox-dependent mechanism of baseplate assembly and tail sheath contraction. These findings establish a minimal myophage that might become a user-friendly chassis phage in synthetic biology.

[DNA mapping in the capsid of giant bacteriophage phiEL (Caudovirales: Myoviridae: Elvirus) by analytical electron microscopy].

INTRODUCTION: Giant phiKZ-like bacteriophages have a unique protein formation inside the capsid, an inner body (IB) with supercoiled DNA molecule wrapped around it. Standard cryo-electron microscopy (cryo-EM) approaches do not allow to distinguish this structure from the surrounding nucleic acid of the phage. We previously developed an analytical approach to visualize protein-DNA complexes on Escherichia coli bacterial cell slices using the chemical element phosphorus as a marker. In the study presented, we adapted this technique for much smaller objects, namely the capsids of phiKZ-like bacteriophages. MATERIAL AND METHODS: Following electron microscopy techniques were used in the study: analytical (AEM) (electron energy loss spectroscopy, EELS), and cryo-EM (images of samples subjected to low and high dose of electron irradiation were compared). RESULTS: We studied DNA packaging inside the capsids of giant bacteriophages phiEL from the Myoviridae family that infect Pseudomonas aeruginosa. Phosphorus distribution maps were obtained, showing an asymmetrical arrangement of DNA inside the capsid. DISCUSSION: We developed and applied an IB imaging technique using a high angle dark-field detector (HAADF) and the STEM-EELS analytical approach. Phosphorus mapping by EELS and cryo-electron microscopy revealed a protein formation as IB within the phage phiEL capsid. The size of IB was estimated using theoretical calculations. CONCLUSION: The developed technique can be applied to study the distribution of phosphorus in other DNA- or RNA-containing viruses at relatively low concentrations of the element sought.

Two Phages of the Genera Felixounavirus Subjected to 12 Hour Challenge on Salmonella Infantis Showed Distinct Genotypic and Phenotypic Changes.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2.

The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the development of alternative antibacterial strategies. Phage therapy has been proven successful for the treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological, genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was 94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were identified for each phage by mass spectrometry and were used to substantiate the function of the predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low multiplicity of infection levels and had good performance both on preventing biofilm formation and eradicating preformed biofilms. They were also stable over a wide range of temperature and pH values, supporting their potential use in the treatment of P. aeruginosa infections.

Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens.

The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called "post-antibiotic era" in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 µg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development.

The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3.

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10⁻1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.

Jet nebulization of bacteriophages with different tail morphologies - Structural effects.

It was previously demonstrated that the loss of infectivity of a myovirus PEV44 after jet nebulization was closely related to a change in bacteriophage (phage) structure. In this follow-up study, we further examined the impact of jet nebulization on tailed phages, which constitute 96% of all known phages, from three different families, Podoviridae (PEV2), Myoviridae (PEV40) andSiphoviridae (D29). Transmission electron microscopy (TEM) identified major changes in phage structures after jet nebulization, correlating with their loss of infectivity. For the podovirus PEV2, jet nebulization had a negligible impact on its activity (0.04 log10 pfu/mL loss) and structural change. On the other hand, the proportion of intact phages in the nebulized samples dropped from 50% to ∼27% for PEV40 and from 15% to ∼2% for D29. Phage deactivation of PEV40 measured by the TEM structural damage (0.52 log10 pfu/mL) was lower than that obtained by plaque assay (1.02 log10 pfu/mL), but within the range of variation (±0.5 log10 pfu/mL). However, TEM quantification considerably underestimated the titer reduction of D29 phage, ∼2 log pfu/mL lower than that obtained in plaque assay (3.25 log10 pfu/mL loss). In conclusion, nebulization-induced titre loss was correlated with morphological damage to phages and in particular, the tail length may be an important consideration for selection of phages in inhaled therapy using jet nebulization.

Structural analysis and proteomics studies on the Myoviridae vibriophage M4.

Bacteriophages play a crucial role in tracking the spread of bacterial epidemics. The frequent emergence of antibiotic-resistant bacterial strains throughout the world has motivated studies on bacteriophages that can potentially be used in phage therapy as an alternative to conventional antibiotic treatment. A recent outbreak of cholera in Haiti took many lives due to a rapid development of resistance to the available antibiotics. The properties of vibriophages, bacteriophages that infect Vibrio cholerae, are therefore of practical interest. A detailed understanding of the structure and assembly of a vibriophage is potentially useful in developing phage therapy against cholera as well as for fabricating artificial nanocontainers. Therefore, the aim of the present study was to determine the three-dimensional organization of vibriophage M4 at sub-nanometer resolution by electron microscopy and single-particle analysis techniques to facilitate its use as a therapeutic agent. We found that M4 has a large capsid with T = 13 icosahedral symmetry and a long contractile tail. This double-stranded DNA phage also contains a head-to-tail connector protein complex that joins the capsid to the tail and a prominent baseplate at the end of the tail. This study also provides information regarding the proteome of this phage, which is proteins similar to that of other Myoviridae phages, and most of the encoded proteins are structural proteins that form the exquisite architecture of this bacteriophage.

High precision microfluidic microencapsulation of bacteriophages for enteric delivery.

A Salmonella specific bacteriophage Felix O1 (Myoviridae) was microencapsulated in a pH responsive polymer formulation. The formulation incorporated a pH responsive methacrylic acid copolymer Eudragit® S100 (10% (w/v)) with the addition of the biopolymer sodium alginate, the composition of which was varied in the range (0.5% (w/v)-2% (w/v)). The microencapsulation process employed commercially available microfluidic droplet generation devices. We have used readily available low cost microfluidic chips instead of bespoke in-house fabricated glass capillary devices which are accessible only in specialist research facilities. We show that these co-flow microfluidic devices can easily be used to prepare phage encapsulated microparticles making them suitable for use by both the phage research community and industry in order to evaluate and optimise phage compatible formulations for microencapsulation. A novelty of the work reported here is that the size of the generated monodispersed droplets could be precisely controlled in the range 50 µm-200 µm by varying the flow rates of the dispersed and continuous phases. Consequently, alginate concentration and microparticle size were shown to influence the phage release profile and the degree of acid protection afforded to phages upon exposure to simulated gastric fluid (SGF). Bigger microparticles (∼100 µm) showed better acid protection compared with smaller beads (∼50 µm) made from the same formulation. Increasing the alginate composition resulted in improved acid protection of phages for similar particle sizes. The high viscosity formulations containing higher amounts of alginate (e.g. 2% (w/v)) negatively affected ease of droplet generation in the microfluidic device thereby posing a limitation in terms of process scale-up. Felix O1 encapsulated in the formulation containing 10% (w/v) ES100 and 1% (w/v) alginate showed excellent protection upon exposure of the gelled microparticles to SGF (pH 1 for 2 h) without the use of any antacids in the encapsulation matrix. Encapsulated phages previously exposed to SGF (pH 1 for 2 h) were released at elevated pH in simulated intestinal fluid (SIF) and were shown to arrest bacterial growth in the log growth phase. We have therefore demonstrated the microencapsulation of phages using readily available microfluidic chips to produce solid dosage microcapsule forms with a rapid pH triggered release profile suitable for targeted delivery and controlled release in the gastrointestinal tract.

Nebulization effects on structural stability of bacteriophage PEV 44.

Reduced infectivity of phage due to the nebulization process has been reported previously, but no visual evidence on structural change upon nebulization has been established, or whether these structural changes can be attributed to the titer reduction. In this study, transmission electron microscopy (TEM) was employed to assess the impact of three different types of nebulizers, air-jet, vibrating-mesh and static-mesh nebulizers, on the structural stability of a Myoviridae phage, PEV44, active against Pseudomonas aeruginosa. The morphology of the phage in the nebulized samples was categorized into two groups: "whole" (the capsid and tail held together) and "broken" (the capsid separated from the tail) phages. The "whole" phage group was further divided into three sub-groups: (1) intact, (2) contracted tail and (3) empty capsid. The starting stock suspension was found to contain considerable portions of "broken" phages (35.5 ±â€¯6.7%), "whole" phages with contracted tails (9.9 ±â€¯5.4%) and empty capsids (19.3 ±â€¯8.9%). The fraction of "broken" phages was significantly increased after nebulization, with the air-jet nebulizer (83%) being more pronounced than the mesh type nebulizers (50-60%). While the amount of intact phages (2-10%) and whole phages with contracted tails (0-3%) were significantly decreased, the proportion of phages with empty capsids (15-36%) were not significantly different. Phages with broken features obtained by TEM quantification were 92.9 ±â€¯3.2%, 74.8 ±â€¯10.4% and 71.2 ±â€¯11.0% for the jet, vibrating-mesh and static-mesh nebulizers, respectively. These results were found to be comparable with the titer loss obtained by the conventional plaque assay results. The in vitro aerosol performance and viable phage delivery of the three nebulizers was also assessed. The Omron nebulizer achieved a significantly higher viable respirable fraction (VRF) than the SideStream and Aeroneb Go (15.1 ±â€¯5.8%, 2.4 ±â€¯2.0%, 4.1 ±â€¯2.7% respectively). In conclusion, this study identified various changes in the phage structure and viability of phage from different types of nebulizers. Understanding these effects and the phage tolerance to nebulization stresses can potentially improve our choice of the delivery method for inhaled phage therapy.

Phage Tail-Like Bacteriocins.

Many dsDNA bacterial viruses (bacteriophages/phages) have long tail structures that serve as organelles for DNA delivery to host targets. These structures, particularly those of Myoviridae and Siphoviridae phages, have an evolutionary relationship with other cellular biological entities that share the common function of penetrating the bacterial envelope. Among these are type VI secretion systems, insecticidal protein complexes, and bacteriocins. Phage tail-like bacteriocins (PTLBs) are widespread in bacteria, comprising different types that likely evolved independently. They can be divided into two major classes: the R-type PTLBs, which are related to contractile Myoviridae phage tails, and the F-type PTLBs, which are related to noncontractile Siphoviridae phage tails. This review provides an overview of the history, biology, and diversity of these entities and also covers recent efforts to utilize these potent bactericidal agents as human therapeutics against bacterial disease.

Characterization of antimicrobial properties of Salmonella phage Felix O1 and Listeria phage A511 embedded in xanthan coatings on Poly(lactic acid) films.

Beyond simply providing a barrier between food and external contaminants, active packaging technologies aim to inhibit pathogen survival and growth within the packaged environment. Bacteriophages have a proven track record as targeted antimicrobials but have yet to be successfully integrated in active packaging without serious loss of activity. We have developed two bacteriophage based xanthan coatings on poly(lactic acid) (PLA) film which significantly inhibits Salmonella Typhimurium and Listeria monocytogenes growth in culture (P < 0.01), and significantly reduces survival and growth of diverse cocktails of Salmonella sp. and L. monocytogenes respectively on precooked sliced turkey breast over 30 days of anaerobic packaging at 4 or 10 °C (P < 0.05). Specifically reductions of 0.832 log at 4 °C and 1.30 log at 10 °C for Salmonella sp., and 6.31 log at 4 °C and 1.52 log at 10 °C for L. monocytogenes were observed. The coating containing Listeria phage A511 also significantly inhibited growth of L. monocytogenes over 14 days in aerobic packaging (3.79 log at 4 °C, 2.17 log at 10 °C, P < 0.05). These coatings showed 99.99% phage release within 30 min for both phages. Similar approaches could be used to develop packaging inhibitory to other significant foodborne pathogens such as Campylobacter, and Escherichia coli, as well as spoilage bacteria.

Incomplete LPS Core-Specific Felix01-Like Virus vB_EcoM_VpaE1.

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics.

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 first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.

Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg(2+) single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfide-bridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.

Identification of the major proteins of the virions of bacteriophage ZF40 Pectobacterium carotovorum.

The vast variety of bacteriophages and the uniqueness of their individual representatives dictate to perform the detailed study of the actual phage-cell interactions, the virion morphogenesis and morphopoiesis in particular. An analysis of the complete genome sequence of the temperate phage ZF40 Pectobacterium carotovorum has shown that it is a representative of a unique group of phages of the Myoviridae family [Comeau A. M, Tremblay D., Moineau S., Rattei T., Kushkina A. I, Tovkach F I., H.M. Krisch, H.W. Ackermann Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses // PLoS ONE.--July 2012. - 7. - N 7. - e40102]. Characteristic features of these viruses are a small length of the tail compared with the diameter of the capsid and a complicated pattern of the tail sheath, leading to its criss-cross striation. In the presented article the major proteins were identified by means of the SDS-PAGE method: the head proteins (mp2: 33.9 kDa), the sheath (mp1: 39.2 kDa) and the tail tube ones (mp3: 19.9 kDa). It was proved that the mp2 molecular weight is the same with the gp46, the putative major capsid protein derived from the results of the genome sequencing. Therefore, it is still not determined whether the gp46 (mp2) of the virulent mutant 421 of the phage ZF40 is exposed to post-translational modification in the course of the phage particle maturation during its development in the cells of the strain M2-4/50RI P. carotovorum. To study the morphogenetic development pathways it was proposed to use the phage variants that form an excess of individual components of the virion: capsids, procapsids and separate tails propagated on different hosts.

Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations.

Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.

Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giant Pseudomonas aeruginosa phage φKZ.

Encased within the 280 kb genome in the capsid of the giant myovirus φKZ is an unusual cylindrical proteinaceous 'inner body' of highly ordered structure. We present here mass spectrometry, bioinformatic and biochemical studies that reveal novel information about the φKZ head and the complex inner body. The identification of 39 cleavage sites in 19 φKZ head proteins indicates cleavage of many prohead proteins forms a major morphogenetic step in φKZ head maturation. The φKZ head protease, gp175, is newly identified here by a bioinformatics approach, as confirmed by a protein expression assay. Gp175 is distantly related to T4 gp21 and recognizes and cleaves head precursors at related but distinct S/A/G-X-E recognition sites. Within the φKZ head there are six high-copy-number proteins that are probable major components of the inner body. The molecular weights of five of these proteins are reduced 35-65% by cleavages making their mature form similar (26-31 kDa), while their precursors are dissimilar (36-88 kDa). Together the six abundant proteins sum to the estimated mass of the inner body (15-20 MDa). The identification of these proteins is important for future studies on the composition and function of the inner body.

The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features.

BACKGROUND: Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies. METHODS: vB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry. RESULTS AND CONCLUSIONS: The DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and ϕ29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle. CONCLUSIONS: Genes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS) degradation, which has not been reported before for Campylobacter phages.