[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.

[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.

[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.

[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.

[Genome conservatism of phiKMV-like bacteriophages (T7 supergroup) active against Pseudomonas aeruginosa].

The T7-like phiKMV bacteriophage active on Pseudomonas aeruginosa was previously isolated by us and shown to have DNA resistant to many endonucleases. A loss of sensitive sites might be a consequence of a long phiKMV evolution on different hosts. To elucidate, whether this trait is shared by other similar phages, several new phiKMV-like phages were isolated from different sources and compared. All studied phiKMV-like phages formed three groups, insignificantly differing in the number and localization of endonuclease-sensitive DNA sites. This confirms that the present-day phages of this species have highly conserved genomes. Mutational "restoration" of the lost sites may be restricted by a lethal effect. The phiKMV-like phages were shown for the first time to increase the rate of in vitro accumulation of giant phiKZ-like phages of P. aeruginosa. This effect is characteristic only of phiKMV-like phages.

[Comparison of genomes of new gigantic Pseudomonas aeruginosa phages from native populations from different regions]. / Sravnenie genomov novykh gigantskikh fagov Pseudomonas aeruginosa iz priorodnykh populiatsii raznykh regionov.

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant phi KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (phi KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all phi KZ-like phages. Phages of the phi KZ species proved to be common in natural populations of various regions, while IL- and Lin68-related phages were extremely rare. Most phi KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from phi KZ may be used to study the origin of phi KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).

[Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa]. / Fenogeneticheskaia kharakteristika gruppy gigantskikh Phi KZ-podobnykh bakteriofagov Pseudomonas aeruginosa.

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.

[Isolation and comparative study of a group of temperate bacteriophages of rhizospheric pseudomonads Pseudomonas putida]. / Vydelenie i sravnitel'noe izuchenie gruppy umerennykh bakteriophagov rizosfernykh psevdomonad Pseudomonas putida.

We have isolated several new temperate bacteriophages for rhizosphere pseudomonads Pseudomonas putida. Examination of these phages, along with two previously isolated temperate phages PP56 and PP71 of P. putida PpG1 (biovar A), allowed us to classify them into four species on the basis of DNA cross-homology; relative genomic size; and, to a certain extent, the morphology of phage particles. Two of these species are represented by nonidentical variants. No transposable phages were found among these two new species. Three phage species cause various-types of lysogenic conversion manifested in growth suppression of other phage species. This seems to account for the fact that the temperate phage of rhizosphere pseudomonads are seldom encountered. The new phages described can be used for selection of phage-resistant bacterial forms exhibiting antifungal activity that are commercially produced and used for treatment of seeds of cultivated plants.

[Comparison of the frequency of imi-r resistant Pseudomonas aeruginosa mutants generated by infection of the bacteria with various bacteriophage-transposons]. / Sravnenie chastot imi-r-ustoichivykh mutantov Pseudomonas aeruginosa, voznikaiushchikh plsle infektsii bakterii razlichnymi bakteriofagami-transpozonami.

We compared the frequencies of imipenem-resistant (imi-r) mutants of a Pseudomonas aeruginosa laboratory strain PAO1 infected by the phages-transposons (PT) specific for this pseudomonad species. The frequency of imi-r mutants among the lysogenic bacteria that appeared after infection reflects the frequency of integrative (conserved) PT transposition into ompD2 gene responsible for synthesis of porin, the protein required for the passage of antibiotic through the cell membrane. After infection by either PT the proportion of imi-r mutants among the lysogenic bacteria was higher than that of spontaneous mutants. The imi-r mutants induced by PT infection form colonies that differ in morphology when grown on different media. The frequencies of imi-r mutants induced by all PT are similar, except for HW12, PM57, and PM62 assigned to a species of the group B3. The phages of this species induce imi-r mutants at a high frequency. Variations in frequencies and colony morphology of imi-r mutants are discussed.

[x811--mutation of the Pseudomonas aeruginosa transposable phage D3112 with a pleiotropic effect]. / x811--mutatsiia faga-transpozona D3112 Pseudomonas aeruginosa s pleiotropnym éffektom.

Characteristics of Pseudomonas aeruginosa Transposable Phage D3112 carrying mutation x811 are described. x811 is a recessive mutation with pleiotropic effect. It determines a deteriorated lysis of infected or induced bacteria, a delayed replication, and a considerably decreased replication rate. In addition, the x811 mutation is expressed as the Kil phenotype, since high-temperature induction of prophage D3112 cts15 x811 does not cause an immediate decrease in the ability of bacteria to form colonies at 42 degrees C. Restriction analysis of DNA of D3112 cts15 x811 and its segregants has not revealed extended insertions or deletions. The characteristics of segregants of the D3112 cts15 x811 phage agree with the suggestion that the x811 mutation has emerged in a regulatory element (a gene or a site) that controls both expression of the entire early operon, including the "pre-early" function Kil, and the regulation of the repressor synthesis.

[Mucoid clones of Pseudomonas aeruginosa PAO1, surviving after induction of prophage transposons]. / Mukoidnye klony Pseudomonas aeruginosa PAO1, vyzhivaiushchie posle induktsii profagov-transpozonov.

The origin and properties of mucoid clones were studied. The clones were selected with high frequency after thermo-induction of Pseudomonas aeruginosa lysogenic for phage transposons (PT). The production of alginate does not promote the survival of lysogenic bacteria at 42 degrees C. Mucoid clones were shown to appear before thermo-induction; the frequency of their formation does not depend on the specificity of the mutator effect intrinsic to different PT. Phenotypic differences typical of mucoid clones can be mediated by different mutations promoting clone survival at 42 degrees C and by simultaneously arising additional mutations. The SL21 mucoid clone selected among clones of P. aeruginosa PAO1 resistant to PT of B3 possesses an additional trait of phage resistance at 42 degrees C. The presence of D3112 cts 15 prophage has no significant effect on the frequency of SL21 reversion to nonmucoidness. This means that the mutator effect of PT has made a slight contribution to this process. The appearance of mutations promoting the survival of the thermoinducible lysogen SL21 (D3112 cts 15) does not affect the frequency of the loss of mucoidness. Nonmucoid derivatives of SL21 were shown to differ in phage resistance at 42 degrees C and in the extent of the residual mucoidness manifested under specific conditions. Consequently, nonmucoid clones appear as a result of pseudo-reversions. Because some of these pseudo-revertants cannot again be converted to the mucoid form, it is concluded that they carry mutations in genes whose functions are obligatory for the production of alginate.

[Origin and properties of Pseudomonas aeruginosa PAO1 clones surviving after induction of prophages-transposons]. / Proiskhozhdenie i svoistva klonov Pseudomonas aeruginosa PAO1, vyzhivaiushchikh posle induktsii profagov-transpozonov.

Various mutations cancelling the lethal effect of phage lytic development and simultaneous phenotypic modifications were found in rare clones surviving after incubation at 42 degrees C of Pseudomonas aeruginosa (D3112 cts 15), lysogenic for thermoinducible mutant cts 15 of the transposable prophage (TP) D3112. All mutations arose prior to thermal induction. Temperature induction of other bacteriophages (nontransposable) did not lead to selection of bacterial morphological mutants. Therefore, it was concluded that mutagenesis occurred upon the partial (reversible) TP derepression accompanied by coupled replication-transposition of TP, the latter being the direct cause of the mutator effect. Isolation of the P. aeruginosa PAO1 mutant R10 (this mutant is resistant to infection with TP at 42 degrees C) allowed the proper selection and examination of numerous survivors. Comparison of their types derived from lysogens with different prophage location indicated that the number of secondary sites where TP integration is possible without the loss of cell viability is limited. Several transposition events occurred in the history of some survivors (during a repeated or single derepression event). Type D clones, which produce small colonies, are of special interest, because mechanisms underlying the survival of such clones are extremely diverse, and their phenotypes indicate the possibility of stable chromosomal rearrangements in the genome of P. aeruginosa.