Contractile Tail Research Articles

Characterization of two novel lytic bacteriophages for reducing biofilms of zoonotic multidrug-resistant Staphylococcus aureus and controlling their growth in milk

This study targeting the characterization of new lytic phages and studying their impact on Staphylococcus aureus biofilm and growth in milk. Two lytic phages (vB_SauM_ME18 & vB_SauM_ME126) were isolated using 40 Multidrug-Resistant (MDR) S. aureus isolates. Four isolates (10%) were susceptible to phages. Phages were related to Family Myoviridae, had icosahedral heads and long contractile tails. The genome size is 20 kbp for both phages and DNA was digested with hinf I, hind III and hae III. Phages had a latent period (~15 min), rise period (~30 min) and burst size (114 PFU/infected cell for ME18 and 140 PFU/infected cell for ME126). There was a significant reduction in biofilm formation and reduction in the established biofilm biomass in phage treated S. aureus compared with controls (P < 0.05). Phages showed high activity and eliminated MDR S. aureus from ultra-heat-temperature treated milk at 25 °C. However, at 37 °C, ME126 (MOI10) eradicated S. aureus and ME18 (MOI10) reduced the CFU/ml by 87.2% compared with controls (P < 0.05). In essence, lytic phages are promising natural antimicrobials for inhibiting the growth and biofilm of MDR S. aureus in milk, thereby, biocontrol of foodborne pathogens.

Probing the Conformational Change of Contractile Tails using Coarse-Grained Normal Mode Analysis

Significant development in nanotechnology is expected to come from understanding the mechanisms of biomolecular machines. Contractile tails are unique organelles present in certain biomolecular machines which aid in piercing a host membrane and injecting macromolecules inside the host. These tails consist of an inner tail tube with a spring-like sheath wrapped around it. The inner tail tube ends at a spike tip surrounded by a baseplate. During the injection mechanism the sheath undergoes a large conformational from an extended state to a contracted state,thereby pushing the tail tube like a needle through the membrane. This conformational change involves ∼1.3 million atoms and is estimated to be on the millisecond-to-second timescale. Recent efforts have yielded information regarding the structure of the sheath. Conventional methods such as molecular dynamics simulations are not possible for a system of this size. Instead, the collective motions can be observed using the anisotropic network model (ANM), a coarse-grained normal mode analysis method. The ANM model shows that the slowest, global modes are encoded within the sheath's structure. Using these modes we show how it is possible to observe a coarse grained representation of the contraction pathway. Furthermore the work presented could be applied to other large biological structures which undergo conformational changes.

Diversity of Sinorhizobium (Ensifer) meliloti Bacteriophages in the Rhizosphere of Medicago marina: Myoviruses, Filamentous and N4-Like Podovirus.

Using different Sinorhizobium meliloti strains as hosts, we isolated eight new virulent phages from the rhizosphere of the coastal legume Medicago marina. Half of the isolated phages showed a very narrow host range while the other half exhibited a wider host range within the strains tested. Electron microscopy studies showed that phages M_ort18, M_sf1.2, and M_sf3.33 belonged to the Myoviridae family with feature long, contractile tails and icosaedral head. Phages I_sf3.21 and I_sf3.10T appeared to have filamentous shape and produced turbid plaques, which is a characteristic of phages from the Inoviridae family. Phage P_ort11 is a member of the Podoviridae, with an icosahedral head and a short tail and was selected for further characterization and genome sequencing. P_ort11 contained linear, double-stranded DNA with a length of 75239 bp and 103 putative open reading frames. BLASTP analysis revealed strong similarities to Escherichia phage N4 and other N4-like phages. This is the first report of filamentous and N4-like phages that infect S. meliloti.

Characteristics of two myoviruses induced from the coastal photoheterotrophic bacterium Porphyrobacter sp. YT40.

In this study, we characterized two induced myoviruses from one marine photoheterotrophic bacterium Porphyrobacter sp. YT40 belonging to the Sphingomonadales family in Alphaproteobacteria. The genome sequence of prophage A is ∼36.9kb with an average GC content of 67.1%, and its core or functional genes are homologous to Mu or Mu-like phages. Furthermore, induced viral particles from prophage A show a knob-like neck structure, which is only found in bacteriophage Mu. The genome size of prophage B is ∼36.8kb with an average GC content of 65.3%. Prophage B contains a conserved gene cluster Q-P-O-N-M-L, which is unique in P2 phages. Induced viral particles from prophage B display an icosahedral head with a diameter of ∼55nm and a 130±5nm long contractile tail. To our knowledge, this is the first report that characterizes the induced P2-like phage in marine Alphaproteobacteria. Phylogeny analyses suggest that these two types of prophages are commonly found in sequenced bacteria of the Sphingomonadales family. This study sheds light on the ongoing interaction between marine bacteria and phages, and improves our understanding of bacterial genomic plasticity and evolution.

A novel jumbo Tenacibaculum maritimum lytic phage with head-fiber-like appendages.

A novel jumbo bacteriophage (myovirus) is described. The lytic phage of Tenacibaculum maritimum, which is the etiological agent of tenacibaculosis in a variety of farmed marine fish worldwide, was plaque-isolated from seawater around a fish aquaculture field in Japan. The phage had an isometric head 110-120 nm in diameter, from which several 50- to 100-nm-long flexible fiber-like appendages emanate, and a 150-nm-long rigid contractile tail. The full genomes of the two representative phages (PTm1 and PTm5) were 224,680 and 226,876 bp long, respectively, both with 29.7% GC content, and the number of predicted open reading frames (ORFs) was 308 and 306, respectively. The average nucleotide sequence identity between PTm1 and PTm5 was 99.95%, indicating they are quite similar to each other. A genetic relationship was found in 15.0-16.6% of the predicted ORFs among the T. maritimum phages PTm1 and PTm5, the Tenacibaculum spp. phage pT24, and the Sphingomonas paucimobilis phage PAU. Phylogenetic analysis based on the terminase large subunit genes revealed that these four phages (PTm1, PTm5, pT24 and PAU) are more closely related than the other 10 jumbo myoviruses that have similar genome sizes. Transmission electron microscopy observations suggest that the head fibers of the T. maritimum phage function as tentacles to search and recognize the host cell surface to facilitate infection.

Characterization of Lytic Bacteriophages Infecting Multidrug-Resistant Shiga Toxigenic Atypical Escherichia coli O177 Strains Isolated From Cattle Feces.

The increasing incidence of antibiotic resistance and emergence of virulent bacterial pathogens, coupled with a lack of new effective antibiotics, has reignited interest in the use of lytic bacteriophage therapy. The aim of this study was to characterize lytic Escherichia coli O177-specific bacteriophages isolated from cattle feces to determine their potential application as biocontrol agents. A total of 31 lytic E. coli O177-specific bacteriophages were isolated. A large proportion (71%) of these phage isolates produced large plaques while 29% produced small plaques on 0.3% soft agar. Based on different plaque morphologies and clarity and size of plaques, eight phages were selected for further analyses. Spot test and efficiency of plating (EOP) analyses were performed to determine the host range for selected phages. Phage morphotype and growth were analyzed using transmission electron microscopy and the one-step growth curve method. Phages were also assessed for thermal and pH stability. The spot test revealed that all selected phages were capable of infecting different environmental E. coli strains. However, none of the phages infected American Type Culture Collection (ATCC) and environmental Salmonella strains. Furthermore, EOP analysis (range: 0.1–1.0) showed that phages were capable of infecting a wide range of E. coli isolates. Selected phage isolates had a similar morphotype (an icosahedral head and a contractile tail) and were classified under the order Caudovirales, Myoviridae family. The icosahedral heads ranged from 81.2 to 110.77 nm, while the contractile tails ranged from 115.55 to 132.57 nm in size. The phages were found to be still active after 60 min of incubation at 37 and 40°C. Incremental levels of pH induced a quadratic response on stability of all phages. The pH optima for all eight phages ranged between 7.6 and 8.0, while at pH 3.0 all phages were inactive. Phage latent period ranged between 15 and 25 min while burst size ranged from 91 to 522 virion particles [plaque-forming unit (PFU)] per infected cell. These results demonstrate that lytic E. coli O177-specific bacteriophages isolated from cattle feces are highly stable and have the capacity to infect different E. coli strains, traits that make them potential biocontrol agents.

Elastic continuum stiffness of contractile tail sheaths from molecular dynamics simulations.

Contractile tails are key components of the biological nanomachinery involved in cell membrane puncturing, where they provide a means to deliver molecules and ions inside cells. Two intriguing examples of contractile tails are those from bacteriophage T4 and R2-pyocin. Although the two systems are different in terms of biological activity, they share a fascinatingly similar injection mechanism, during which the tail sheaths of both systems contract from a so-called extended state to around half of their length (the contracted state), accompanied by release of elastic energy originally stored in the sheath. Despite the great prevalence and biomedical importance of contractile delivery systems, many fundamental details of their injection machinery and dynamics are still unknown. In this work, we calculate the bending and torsional stiffness constants of a helical tail sheath strand of bacteriophage T4 and R2-pyocin, in both extended and contracted states, using molecular dynamics simulations of about one-sixth of the entire sheath. Differences in stiffness constants between the two systems are rationalized by comparing their all-atom monomer structures, changes in sheath architecture on contraction, and differences in interstrand interactions. The calculated coefficients indicate that the T4 strand is stiffer for both bending and torsion than the corresponding R2-pyocin strands in both extended and contracted conformations. The sheath strands also have greater stiffness in the contracted state for both systems. As the main application of this study, we describe how the stiffness constants can be incorporated in a model to simulate the dynamics of contractile nanoinjection machineries.

Mobile Loops and Electrostatic Interactions Maintain the Flexible Tail Tube of Bacteriophage Lambda

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

Type VI Secretion System in Pathogenic Escherichia coli: Structure, Role in Virulence, and Acquisition.

Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gram-negative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and Escherichia coli is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, E. coli is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell–cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of E. coli pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread.

Atomic structures of an entire contractile injection system in both the extended and contracted states.

Contractile injection systems are sophisticated multiprotein nanomachines that puncture target cell membranes. While the amount of atomic resolution insights into contractile bacteriophage tails, bacterial type six secretion systems and R-pyocins is rapidly increasing, structural information about contraction of bacterial phage-like protein-translocation structures directed towards eukaryotic hosts is scarce. Here we characterise the antifeeding prophage AFP from Serratia entomophila by cryo-electron microscopy. We present the high-resolution structure of the entire AFP particle in the extended state, trace 11 protein chains de novo from the apical cap to the needle tip, describe localisation variants and perform specific structural comparisons with related systems. We analyse intersubunit interactions and highlight their universal conservation within contractile injection systems while revealing the specificities of AFP. Furthermore, we provide the structure of the AFP sheath-baseplate complex in a contracted state. This study reveals atomic details of interaction networks that accompany and define the contraction mechanism of toxin-delivery tailocins, offering a comprehensive framework for understanding their mode of action and for their possible adaptation as biocontrol agents.

Time-resolved DNA release from an O-antigen–specific Salmonella bacteriophage with a contractile tail

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Å resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

A65 Characterization of endolysin gene of bacteriophages infecting Listeria spp. isolated from dairy industry wastewater

Bacteriophages and their endolysins, enzymes that degrade the cell walls of bacteria, are emerging as alternative tools to detect and inhibit growth of pathogen bacteria. Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a serious invasive disease that affects both humans and a wide range of animals. Listeria spp. are ubiquitous in the dairy farm environment and could be present in dairy-processing plants and wastewater. All Listeria-specific bacteriophages found to date are members of the Caudovirales, of the Siphoviridae or Myoviridae families. Myophages infecting Listeria have been recently classified by the ICTV in the Spounavirinae subfamily, as well as in the P100 virus genus. The aim of this work was to isolate Listeria spp. bacteriophages and their endolysin codifying genes from wastewater of a dairy industry. Wastewater with and without treatment was sampled during the course of a year, and isolation of bacteriophages was performed after an enrichment step using as hosts L. innocua, L. ivanovii, and L. monocytogenes serotypes 1/2a, 1/2b, and 4b. Bacteriophages infecting L. innocua and L. ivanovii were isolated (n = 24) from 3 out of 12 samples. Bacteriophages were purified, and the host range was determined using spot test and EOP against five collection strains and several field isolates of Listeria spp. Two bacteriophages of narrow and broad host range, vB_Lino_VEfB7, and vB_Liva_VAfA18, were selected for further characterization. High titer stocks of bacteriophages were purified by centrifugation with ammonium acetate, and morphological information on the purified bacteriophages was obtained by negative staining and transmission electronic microscopy. Their morphology, size, and contractile tails indicated that these bacteriophages belonged to the Myoviridae family. Bacteriophage genomes were extracted using phenol-chloroform, followed by ethanol precipitation, and tested by digestion with RNAsa A and DNAse I. RFLP was performed, digesting genomes with restriction enzymes HindIII and NcoI. Consistent with the morphological findings, bacteriophages contained dsDNA genomes but showed different RFLP patterns. A PCR designed to amplify conserved domains of endolysins—PGRP and CwlA—was applied to characterize this gene. Another PCR was designed to amplify the complete endolysin gene, and the complete sequence of this gene was obtained and analyzed. Substitution model selection and a maximum likelihood phylogenetic tree of the endolysin gene was carried out using IQ-Tree software. The sequences of the endolysin gene indicated that the codified enzyme is an N-acetyl-muramoyl-L-alanine amidase, related to A511 and P100 species of the recently described P100virus genus. Further evolutionary analyses are needed to evaluate their belonging to this species or their taxonomy within this genus.

Characterization and Genome Analysis of a Novel Alteromonas Phage JH01 Isolated from the Qingdao Coast of China.

A novel Alteromonas phage JH01, with the host strain identified to be Alteromonas marina SW-47(T), was isolated from the Qingdao coast during the summer of 2017. Transmission electron microscopy analysis showed that phage JH01 can be categorized into the Siphoviridae family, with an icosahedral head of 62 ± 5nm and a long contractile tail of 254 ± 10nm. The bioinformatic analysis shows that this phage consists of a linear, double-stranded 46,500bp DNA molecule with a GC content of 44.39%, and 58 ORFs with no tRNA genes. The ORFs are classified into four groups, including phage packaging, phage structure, DNA replication and regulation, and hypothetical protein. The phylogenetic tree, constructed using neighbor-joining analysis, shows that phage JH01 has altitudinal homology with some Vibrio and Pseudoalteromonas phage B8b. Comparative analysis reveals the high similarity between phage JH01 and phage B8b. Additionally, our study of phage JH01 provides useful information for further research on the interaction between Alteromonas phages and their hosts.

Delivery of the Pseudomonas aeruginosa Phospholipase Effectors PldA and PldB in a VgrG- and H2-T6SS-Dependent Manner.

The bacterial pathogen Pseudomonas aeruginosa uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which P. aeruginosa produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote vgrG and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called “à la carte delivery.” Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered via their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery.

Structure and Activity of the Type VI Secretion System.

The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells. The injection apparatus is composed of a baseplate on which is built a contractile tail tube/sheath complex. The inner tube, topped by the spike complex, is propelled outside of the cell by the contraction of the sheath. The injection system is anchored to the cell envelope and oriented towards the cell exterior by a trans-envelope complex. Effectors delivered by the T6SS are loaded within the inner tube or on the spike complex and can target prokaryotic and/or eukaryotic cells. Here we summarize the structure, assembly, and mechanism of action of the T6SS. We also review the function of effectors and their mode of recruitment and delivery.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P.asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing ahexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

Molecular and Biological Characterization of Ralstonia Phage RsoM1USA, a New Species of P2virus, Isolated in the United States

The first Ralstonia-infecting bacteriophage from soil of the United States, designated RsoM1USA, was isolated from a tomato field in Florida. Electron microscopy revealed that phage RsoM1USA is member of the genus P2virus in the family Myoviridae with an icosahedral head of about 66 nm in diameter, a contractile tail of about 152 nm in length, and a long “neck.” Phage RsoM1USA infected 12 of the 30 tested R. solanacearum species complex strains collected worldwide in each of the three Ralstonia species: R. solanacearum, R. pseudosolanacearum, and R. syzygii. The phage completed its infection cycle 180 min post infection with a burst size of about 56 particles per cell. Phage RsoM1USA has a genome of 39,309 nucleotides containing 58 open reading frames (ORFs) and is closely related to Ralstonia phage RSA1 of the species Ralstonia virus RSA1. The genomic organization of phage RsoM1USA is also similar to that of phage RSA1, but their integrases share no sequence homology. In addition, we determined that the integration of phage RsoM1USA into its susceptible R. solanacearum strain K60 is mediated by the 3′ 45-base portion of the threonine tRNA (TGT), not arginine tRNA (CCG) as reported for phage RSA1, confirming that the two phages use different mechanism for integration. Our proteomic analysis of the purified virions supported the annotation of the main structural proteins. Infection of a susceptible R. solanacearum strain RUN302 by phage RsoM1USA resulted in significantly reduced growth of the infected bacterium in vitro, but not virulence in tomato plants, as compared to its uninfected RUN302 strain. Due to its differences from phage RSA1, phage RsoM1USA should be considered the type member of a new species with a proposed species name of Ralstonia virus RsoM1USA.

Characterization of a novel lytic myophage, phiA8-29, infecting Aeromonas strains.

Members of the bacterial genus Aeromonas are important aquatic pathogens that cause severe fish diseases. Here, we characterize a novel lytic phage, Aeromonas virus phiA8-29, isolated from the alimentary tract of a freshwater fish. Transmission electron microscopy showed that phiA8-29 has a long contractile tail and thus can be classified as a member of the family Myoviridae. The phage genome was identified as a double-stranded DNA molecule of 144,974 bp containing 185 open reading frames and nine tRNA-encoding genes. Comparative genomic analysis revealed that the phiA8-29 genome has little similarity to any of the currently sequenced Aeromonas phage genomes. Our data indicate that phiA8-29 is a novel lytic Myoviridae phage that does not belong to any of the known genera.

Isolation and Characterization of a Lytic and Highly Specific Phage against Yersinia enterocolitica as a Novel Biocontrol Agent.

The aim of this study was to isolate and characterize a lytic Yersinia enterocolitica-specific phage (KFS-YE) as a biocontrol agent. KFS-YE was isolated and purified with the final concentration of (11.72 ± 0.03) log PFU/ml from poultry. As observed by transmission electron microscopy, KFS-YE consisted of an icosahedral head and a contractile tail, and was classified in the Myoviridae family. KFS-YE showed excellent narrow specificity against Y. enterocolitica only. Its lytic activity was stable at wide ranges of pH (4-11) and temperature (4-50°C). The latent period and burst size of KFS-YE were determined to be 45 min and 38 PFU/cell, respectively. KFS-YE showed relatively robust storage stability at -20, 4, and 22°C for 40 weeks. KFS-YE demonstrated a bactericidal effect in vitro against Y. enterocolitica and provided excellent efficiency with a multiplicity of infection as low as 0.01. This study demonstrated the excellent specificity, stability, and efficacy of KFS-YE as a novel biocontrol agent. KFS-YE may be employed as a practical and promising biocontrol agent against Y. enterocolitica in food.