The Bacteriophage pEp_SNUABM_08 Is a Novel Singleton Siphovirus with High Host Specificity for Erwinia pyrifoliae.

Species belonging to the genus Erwinia are predominantly plant pathogens. A number of bacteriophages capable of infecting Erwinia have been used for the control of plant diseases such as fire blight. Public repositories provide the complete genome information for such phages, which includes genomes ranging from 30 kb to 350 kb in size. However, limited information is available regarding bacteriophages belonging to the family Siphoviridae. A novel lytic siphophage, pEp_SNUABM_08, which specifically infects Erwinia pyrifoliae, was isolated from the soil of an affected apple orchard in South Korea. A comprehensive genome analysis was performed using the Erwinia-infecting siphophage. The whole genome of pEp_SNUABM_08 comprised 62,784 bp (GC content, 57.24%) with 79 open reading frames. The genomic characteristics confirmed that pEp_SNUABM_08 is a singleton lytic bacteriophage belonging to the family Siphoviridae, and no closely related phages have been reported thus far. Our study not only characterized a unique phage, but also provides insight into the genetic diversity of Erwinia bacteriophages.

Genomic comparison of 60 completely sequenced bacteriophages that infect Erwinia and/or Pantoea bacteria.

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.

Complete genome sequence analysis of temperate Erwinia bacteriophages 49 and 59.

To date, a small number of temperate phages are known to infect members of the genus Erwinia. In this study, the genomes of temperate phages vB_EhrS_49 and vB_EhrS_59 infecting Erwinia horticola, the causative agent of beech black bacteriosis in Ukraine, were sequenced and annotated. Their genomes reveal no significant similarity to that of any previously reported viruses of Enterobacteriaceae. At the same time, phages 49 and 59 share extensive nucleotide sequence identity across the regions encoding head assembly, DNA packaging, and lysis. Despite significant homology between structural modules, the organization of distal tail morphogenesis genes is different. Furthermore, a number of putative morons and DNA methylases have been found in both phage genomes. Due to the revealed synteny as well as the structure of lysogeny module, phages 49 and 59 are suggested to be novel members of the lambdoid phage group. Conservative structural genes together with varying homology across the nonstructural region of the genomes make phages 49 and 59 highly promising objects for studying the genetic recombination and evolution of microbial viruses. The obtained data may as well be helpful for better understanding of relationships among Erwinia species.

Characterization of two related Erwinia myoviruses that are distant relatives of the PhiKZ-like Jumbo phages.

Bacteriophages are a major force in the evolution of bacteria due to their sheer abundance as well as their ability to infect and kill their hosts and to transfer genetic material. Bacteriophages that infect the Enterobacteriaceae family are of particular interest because this bacterial family contains dangerous animal and plant pathogens. Herein we report the isolation and characterization of two jumbo myovirus Erwinia phages, RisingSun and Joad, collected from apple trees. These two genomes are nearly identical with Joad harboring two additional putative gene products. Despite mass spectrometry data that support the putative annotation, 43% of their gene products have no significant BLASTP hit. These phages are also more closely related to Pseudomonas and Vibrio phages than to published Enterobacteriaceae phages. Of the 140 gene products with a BLASTP hit, 81% and 63% of the closest hits correspond to gene products from Pseudomonas and Vibrio phages, respectively. This relatedness may reflect their ecological niche, rather than the evolutionary history of their host. Despite the presence of over 800 Enterobacteriaceae phages on NCBI, the uniqueness of these two phages highlights the diversity of Enterobacteriaceae phages still to be discovered.

MORPHOLOGICAL HETEROGENEITY OF TEMPERATE ERWINIOPHAGE 59.

This paper is devoted to the phenomenon of morphological heterogeneity within the population of the temperate erwiniophage 59, which does not have analogies among other bacterial viruses. AIM: To investigate the basic properties of erwiniophage 59 heteroge- neous population obtained from different isogenic strains of amilovora-like bacterium Erwinia "horticola" (Eho). METHODS: Erwiniophage 59 was obtained by propagation on its traditional host Eho 450, as well as on its three isogenic strains and a related bacterium E. "horticola" 60. Physical and chemical properties of the phage particles were studied using centrifugation in CsCl-gradients, electrophoresis in agarose gels, electron micros- copy, restriction analysis ofDNA and SDS-PA GE of the virion polypeptides. RESULTS: It was shown that the pool oftemperatephage 59 is a heterogeneous population consisting of two phage types when propagated on the mentioned hosts. These types are discrete and have buoyant-density difference of 0.02 g/cm3, that allowed to separate subpopulations for a de- tailed investigation. The subpopulation with the higher density was determined as the au- thentic bacteriophage 59 (subpopulation II with capsid diameter of55.36 nm). The capsid diameter of the subpopulation Iparticles equals 51.16 nm. Both types ofparticles do not differ by DNA size and have identical restriction patterns. Based on the SmaI-restriction analysis it may be concluded that the DNA packaging remains unchangeable and is carried out according to the headfull packaging mechanism. However; the subpopulation I differs from the original one by a relative content of some polypeptides. Curiously, the subpopulations I and II have different values of lysogenization and spontaneous induction frequencies. CONCLUSIONS: An unusual type of morphological heterogeneity ofthe phage 59 particles was observedfor thefirst time; this heterogeneity is associated with the presence of two equimolar subpopulations with different physical and chemical parametrs of the virions. Morphological heterogeneity of temperate erwiniophage 59 significantly differs from such of the classical coliphages as T4, P1 and the phage system P2-P4.

Exclusion of polyvalent T7-like phages by prophage elements.

The study presents new insights into the process of interaction of T7-like bacteriophages FE44 and BA14 with lysogenic cells. It was demonstrated that single and double lysogens possess Abiphenotype regardless of genera, species and strain of bacteria that initially had normal phage sensitivity. Efficiency of plating of these phages is reduced by two orders of magnitude on monolysogens, whereas it decreases by 4-6 orders on bilysogens. In the latter case, phage infection leads to formation of more than 60% of aberrant capsids in phage progeny. Abortive phage infection is suggested to be associated with defects in general dynamics of the bacterial chromosome in single and double lysogens of Erwinia "horticola" and Escherichia coli.

Phytopathogenic bacteria phenotype conversion as a result of their lysogenisation by coliphage P1.

A set of lysogenic strains of phytopathogenic bacteria Erwinia "horticola" and Erwinia amylovora associated with woody plants was obtained using bacteriophage P1 Cmc1ts100. The phenotype conversion from Cm(S) to Cm(R) was shown to be connected with introducing of authentic prophage DNA of 94.8 kb as a single-copy plasmid into the cells. Prophage state is unstable: P1 plasmid is spontaneously lost with high frequency by the cells. In lysogenic cells the prophage genes of type III restriction-modification complex EcoP1I are actively expressed. The system formed by E. "horticola" 450 and 60 as well as their lysogenic derivatives and specific bacteriophages provides an opportunity to divide the latter into three groups according to the level of restriction in the course of their interaction with the enzyme EcoP1I. The difference in phage responses to the endonuclease presence in a lysogenized host presumably correlates with the number of enzyme recognition sequences and the adsorption sites availability. After the prophage plasmid DNA curing the characteristic value of phage sensitivity of cells is changed. The lysogenic strains obtained in this work allow for the exploration of EcoP1I restriction-modification gene complex interaction with polyvalent phages able to grow not only on E. coli, but also on such phytopathogens as E. "horticola" and E. amylovora.

Tasmancin and lysogenic bacteriophages induced from Erwinia tasmaniensis strains.

Mitomycin C treatment of Erwinia tasmaniensis strains from Australia induced prophages and the expression of bacteriocins. The bacteriocin named tasmancin inhibited E. tasmaniensis strains from South Africa and Germany. A gene cluster with a klebicin-related operon and an immunity protein was detected on plasmid pET46 from E. tasmaniensis strain Et1/99. PCR reactions using primers directed to this region produced signals for several strains originating from Australia, but not for strains isolated in South Africa and Germany. The latter isolates lacked plasmid pET46. Bacteriophages were induced from E. tasmaniensis strains Et88 and Et14/99, both isolates from South-Eastern Australia. These phages formed plaques on several other strains from this region, as well as on E. tasmaniensis strains from South Africa and Germany. Sequencing revealed similarity of phages ϕEt88 and ϕEt14, which shared the host range on E. tasmaniensis strains. Bacteriophages and tasmancin may interfere with the viability of several related E. tasmaniensis strains in the environment of carrier strains.

Complete genome sequences of three Erwinia amylovora phages isolated in north america and a bacteriophage induced from an Erwinia tasmaniensis strain.

Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.

Mutagenesis and functional characterization of the RNA and protein components of the toxIN abortive infection and toxin-antitoxin locus of Erwinia.

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

[New tendencies in molecular genetics of bacteriophages].

The paper deals with development of molecular genetics of bacteriophages of phytopathogenic erwinia at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine.

Bacteriophages of Erwinia amylovora.

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:

[Comparative study of properties of temperate erwiniophages 49 and 59]. / Sravnitel'noe izuchenie svoistv umerennykh érviniofagov 49 i 59.

Molecular-biological properties of two relative temperate erwiniophages 49 and 59 have been comparatively studied. The both phages are highly specific with respect to sensitive bacteria and lyse only inconsiderable quantity of amylovora-like strains of Erwinia horticola. It has been established that erwiniophages are distinguished by the basic parameters of a single reproduction cycle in the cells of common host E. horticola 450. Considerable differences between phages have been also found in the areas of genomes responsible for the establishment and maintenance of lysogenic state in the cells of the bacterium-host. Study of structure polypeptides has confirmed the identity of capsids and tails of phages 49 and 59. It has been shown that phage 49 has another, as compared to phage 59, basal plate, which availability destabilises the phage tail and leads to virion destruction under various physical effects. Virion DNA of phages 49 and 59 are of the same size--47.9 kbp, but differ as to GC-content. Using the restriction analysis it has been shown that genome of phage 49, as well as the genome of phage 59, is permuted, but its permutation is of discrete character. The fact of recombination interaction between erwiniophages 49 and 59 has been established. It is supposed that phage 49 is the recombination (hybrid) derivative of phage 59 and unknown phage, or prophage, genetic module. The given recombination, probably, took place under the persistence of different phages in the general polylysogenic system of E. horticola.

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora.

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.