[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.

[NOVEL APPROACH TO COMPOSITION OF, BACTERIOPHAGE MIXTURES FOR ANTIBACTERIAL THERAPY].

AIM: Evaluate antibacterial activity of an experimental mixture of phages, belonging to several well-studied species. MATERIALS AND METHODS: The study was carried out using a group of 55 clinical Pseudomonas aeruginosa strains of various origins,- 4 mono-species mixtures of 32 virulent bacteriophages (species phiKZ-, phiKMV-, phiPBl-, PaP3-like phages) and 2 novel phages, phiMK (species PaK-P2) and phiPerm5. Activity of preparations from mono-species mixtures of bacteriophages ofvarious species were compared with activity of 3 commercial mixtures. Standard methods of study of bacteriophages were used: determination of lytic activity by seeding onto bacterial lawns of P. aeruginosa, restriction analysis of phage DNA for confirmation of their be- longing to certain species. RESULTS: Cumulative activity of 6 mono-species mixtures of virulent phages was shown to be similar to lytic activity of commercial therapeutic mixtures used against P. aeruginosa infections. 54 of 55 strains of clinical isolates of P: aeruginosa showed sensitivity to experimental mixtures composed of mono-species mixtures of bacteriophages. 53 strains were lysed by commercial preparations. Wherein the possibility of accidental inclusion of moderate -bacteriophages in the experimental mixture is excluded. CONCLUSION: A possibility of creation of highly active therapeutic antibacterial preparations against P. aeruginosa using mono-species mixtures of 6 species of lytic bacteriophages is shown Use of such a mixture in therapy of lung infections reduces the risk of emergence of bacterial strains with increased virulence and patho- genicity during prolonged administration.

Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa.

A unique feature of the Pseudomonas aeruginosa giant phage phiKZ is its way of genome packaging onto a spool-like protein structure, the inner body. Until recently, no similar structures have been detected in other phages. We have studied DNA packaging in P. aeruginosa phages EL and Lin68 using cryo-electron microscopy and revealed the presence of inner bodies. The shape and positioning of the inner body and the density of the DNA packaging in EL are different from those found in phiKZ and Lin68. This internal organization explains how the shorter EL genome is packed into a large EL capsid, which has the same external dimensions as the capsids of phiKZ and Lin68. The similarity in the structural organization in EL and other phiKZ-like phages indicates that EL is phylogenetically related to other phiKZ-like phages, and, despite the lack of detectable DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor.

[New temperate Pseudomonas aeruginosa phage, phi297: specific features of genome structure].

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.

[Properties of the new D3-like Pseudomonas aeruginosa bacteriophage phiPMG1: genome structure and prospects for the use in phage therapy].

Results of studying the novel virulent phage phiPMG1 active on Pseudomonas aeruginosa are presented. phiPMG1 was shown to exhibit detectable homology and resemblance in the total genome structure with temperate converting phage D3. Phage phiPMG1 differs from D3 in that it fails to stably lysogenize bacteria and can grow on strains carrying plasmids that cause growth inhibition of phage D3 and some other phages. This significantly diminishes the probability of horizontal gene transfer with phage phiPMG1 and suggests the possible employment of this phage in phage therapy. A comparison of genome structures in phages phiPMG1 and D3 demonstrated not only high homology of 65 genes, but also the presence in the phiPMG1 genome of 16 genes that were not recorded in the files of NCBI database. Apparently, the evolution of genomes in phages of this species is mostly associated with migrations into other species of bacteria and recombinations with phages of other species (for example, F116). Detailed structural analysis a genome region in which the essential nonhomology is exhibited between three D3-like phages (D3, phiPMG1, and PAJU2) revealed that the phiPMG1 genome supposedly is phylogenetically closer than the others to the genome of a hypothetical ancestor phage belonging to this species.

Complete genome sequence of the giant Pseudomonas phage Lu11.

The complete genome sequence of the giant Pseudomonas phage Lu11 was determined, comparing 454 and Sanger sequencing. The double-stranded DNA (dsDNA) genome is 280,538 bp long and encodes 391 open reading frames (ORFs) and no tRNAs. The closest relative is Ralstonia phage ϕRSL1, encoding 40 similar proteins. As such, Lu11 can be considered phylogenetically unique within the Myoviridae and indicates the diversity of the giant phages within this family.

[Bacteriophage phi297--the new species of temperate phages Pseudomonas aeruginosa with a mosaic genome: potential use in phagotherapy].

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi97 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable oflysing bacteria, while the wild-type phage induced lysogeny, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm'). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lystic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.

[Genome instability of Pseudomonas aeruginosa phages of the EL species: examination of virulent mutants].

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.

[TL, the new bacteriophage of Pseudomonas aeruginosa and its application for the search of halo-producing bacteriophages].

The properties of new virulent bacteriophage TL of Pseudomonas aeruginosa belonging to the family Podoviridae (genome size of 46 kb) were investigated. This bacteriophage is capable of lysogenizing the bacterial lawn in halo zones around negative colonies (NC) of other bacteriophages. TL forms large NC, that are hardly distinguishable on the lawn of P. aeruginisa PAO1. At the same time, on the lawns of some phage-resistant PAO1 mutants, as well as on those produced by a number of clinical isolates, TL forms more transparent NC. It is suggested that more effective growth of the bacteriophage TL NC is associated with the differences in outer lipopolysaccharide (LPS) layer of the cell walls of different bacterial strains, as well as of the bacteria inside and outside of the halos. This TL property was used to optimize selection of bacteriophages producing halos around NC on the lawn of P. aeruginosa PAO1. As a result, a group of bacteriophages differing in the patterns of interaction between their halos and TL bacteriophage, as well as in some characters was identified. Taking into consideration the importance of cell-surfaced structures of P. aeruginosa in manifestation of virulence and pathogenicity, possible utilization of specific phage enzymes, polysacchadide depolymerases, for more effective treatment of P. aeruginosa infections is discussed.

[The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1].

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.

Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain.

We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

[Induction and migration of cryptic/defective Salmonella enterica prophages as a consequence of infection with lytic phages is an additional factor in stability of a coevolutionary vector].

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.

[Interspecific migration and evolution of bacteriophages of the genus phiKZ: the purpose and criteria of the search for new phiKZ-like bacteriophages].

Some properties of bacteriophages with large (200 kb and more) sequenced genomes have been compared. In contrast to other large bacteriophages from different families, bacteriophages active on pseudomonads of various species (phiKZ-like bacteriophages) have some common features, which suggests their phylogenetic relationship and independence of their evolution as a result of migration among bacteria of this family. Among such common features are the absence in the genomes of these phages of sites sensitive to endonuclease PstI, the absence of genes encoding DNA polymerases that are similar to the known enzymes of this type, possible dependence of replication of the phage genome on bacterial DNA polymerase, and a considerably larger average gene size as compared to that for other phages. Criteria are suggested for searching for novel phiKZ-like bacteriophages: the size of a phage particle, production by bacteria infected with such phages of a large amount of highly viscous mucus. Taking into account the use of these bacteriophages in therapeutic preparations (due to a broad spectrum of lytic activity) and a poor knowledge of a majority of their gene products, it seems necessary to perform a more comprehensive genetic analysis of phages of this genus or their mutants for selecting those adequate for phage therapy.

[Pseudolysogeny of Pseudomonas aeruginosa bacteria infected with phiKZ-like bacteriophages].

In this work, a final piece of evidence proving that bacteria Pseudomonas aeruginosa are capable of transition to the pseudolysogenic state after infection with phiKZ-like phages has been produced. It was shown that the decisive factor in this process is multiple infection of bacteria with bacteriophages belonging to this genus. In the course of this work, stable clinical isolates of bacteria liberating novel bacteriophages of this genus (Che2/2 and Che21/5) were detected and attributed to species phiKZ and EL, respectively, according to their phenotypic characters and the results of DNA analysis. For three bacteriophages belonging to species EL (EL, RU, and Che21/5), mutants with disorders in the capability for pseudolysogenization were isolated. One of the mutants of phage EL possesses properties of virulent mutants of typical temperate phages (vir mutant). This mutant fails to form pseudolysogens and, moreover, provides the effect of dominance upon coinfection of bacteria with the wild-type phage EL, but however is unable to exhibit this effect upon joint infection of bacteria with wild-type phages of species phiKZ and Lin68. It is assumed that the effect of pseudolysogeny may be connected with functioning of phiKZ and EL genes that control the products similar to repressors of other phages. Because earlier wild-type phiKZ-like phages were shown to be present in commercial phage-therapeutic preparations (which represents certain problems), it is expedient to use virulent mutants of phages belonging to this genus rather than phages of the wild type.

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry.

AIMS: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. METHODS AND RESULTS: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. CONCLUSIONS: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage-related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.

[Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products].

Comparison of Pseudomonas putida group of phages attributed to five species (af, phi15, phi27, phi2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.

[A formal scheme of adsorptional receptors in Pseudomonas aeruginosa and possibilities for its practical implementation].

In recent years, a revival of interest to study of bacteriophages has been observed. Bacteriophages and phage-coded products can be used as antibiotics in the treatment of some human and animal infections caused by antibiotic-resistant bacterial strains. Bacteriophages may serve as an excellent method for monitoring anthropogenic changes in bacterial communities, which are connected with the contamination by industrial sewages or infection of water reservoirs with pathogenic bacteria. Technical applicability of bacteriophages may be successful for combating bacterial biofilms, for example, in pipelines. And finally, comparative basic and systemic genomic studies of bacteriophages belonging to various bacterial species are decisive in understanding and assessing their role in the joint evolution (coevolution) with host bacteria; particularly, this research is important for elucidating mechanisms of phage participation in the horizontal exchange of genetic modules. Possibly, these studies may be useful for the prediction of not only the direction of coevolution of certain bacterial species and their phages but also the time of novel pathogenic bacteria origination.

[Comparison of DNA sizes in a group of giant Pseudomonas aeruginosa phages by the PFGE method].

Genome sizes of Pseudomonas aeruginosa phages phiKZ and EL earlier determined by sequence analysis were shown to correspond to sizes of their DNAs assessed by pulse-electrophoresis (PFGE). Putative "redundant" genes in phiKZ phage genome are supposed to control functions promoting vigorous growth of the phage belonging to this species, compared to phages of EL species.

[Study of the diversity in a group of phages of Pseudomonas aeruginosa species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants].

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1, These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.

[Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa].

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.