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.

Рассматривается и обсуждается структура генома и свойства умеренного бактериофага phi297 Pseudomonas aeruginosa. Из анализа результатов секвенирования и аннотирования сделан вывод, что геном phi297 обладает мозаичной структурой, которая сформировалась при комбинации блоков генов бактерий и/или их фагов, относящихся к отдаленным в систематическом отношении группам микроорганизмов. Результаты анализа уровня и характера ДНК-гомологии phi297 с секвенированными к настоящему времени геномами родственных бактериофагов P. aeruginosa интерпретируются с позиции возможности активной миграции фагов этой группы между разными видами бактерий.

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.

Приведены результаты изучения нового вирулентного фага phiPMG1, активного на бактериях вида Pseudomonas aeruginosa. Показано, что phiPMG1 проявляет значительную гомологию и сходство в общей структуре генома с умеренным конвертирующим фагом D3. Фаг phiPMG1 отличается от D3 тем, что не способен осуществлять стабильную лизогенизацию и растет на штаммах с плазмидами, подавляющими развитие фага D3 и некоторых других фагов. Это понижает вероятность горизонтального генетического переноса фагом phiPMG и делает возможным его использование в фаготерапии. Сравнение структуры геномов фагов phiPMG1 и D3 выявило не только значительную гомологию 65 генов, но и присутствие в геноме phiPMG1 16 генов, не представленных в базе данных NCBI. Мы предполагаем, что эволюция геномов фагов этого вида в значительной степени связана с миграциями в другие виды бактерий и рекомбинациями с фагами других видов (например, F116). Подробный анализ структуры одной из областей геномов, в которой проявляется существенная негомология у трех D3-подобных фагов (D3, phiPMG1 и PAJU2), показал, что возможно геном phiPMG1 ближе других к геному гипотетического предкового фага этого вида.

Results of studying the novel virulent phage phiPMG1 active on Pseudomonas aeruginosa are presented. It is shown that phiPMG1 exhibits significant homology and the similarity in the overall structure with the genome of a temperate phage converts 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 phages phiPMG1 and D3 structures of genomes in demonstrated not only high homology of 65 genes, but also the presence of 16 genes in the phiPMG1 genome that were not included in the in 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). A detailed analysis of structure of one region genomes, which significant nonhomology for the three D3-like phages (D3, phiPMG1 and PAJU2), revealed that the phiPMG1 genome possible closest to a hypothetical genome of ancestral phage of this species.

Продолжено изучение явления псевдолизогении у бактерий Pseudomonas aeruginosa, инфицированных phiKZ-подобными фагами вида EL. Проведен анализ группы вновь изолированных vir-мутантов фагов вида EL (EL и RU), проявляющих вирулентность (способность лизировать бактерии, зараженные фагом дикого типа) и пониженный уровень опалесценции негативных колоний (НК). Выявление рекомбинантов дикого типа в скрещиваниях vir-мутантов фагов EL и RU подтверждает полигенный контроль этого признака. Делеционный характер мутации у одного из vir-мутантов фага EL, а также высокий уровень генетической нестабильности другого мутанта предполагают возможное участие в мутагенезе мобильного генетического элемента. Возникновение в бактерии псевдолизогенного состояния создает условия для горизонтального переноса генов между разными штаммами бактерий. Таким образом, только секвенирования геномов фагов и доказательства отсутствия у них генопродуктов с токсическим эффектом оказывается недостаточным для включения фагов в лечебные смеси. Обязательным условием использования живых фагов является детальное изучение возможных результатов их взаимодействия с бактериальным хозяином.

Приведены результаты исследования генома умеренного ореолобразующего бактериофага Pseudomonas aeruginosa phi297 и литической активности его вирулентного мутанта. Определение полной последовательности генома этого бактериофага обнаружило его мозаичную структуру. Часть последовательности генома phi297 гомологична последовательности генома фагов D3 и F116. Особенности морфологии негативных колоний (НК) умеренного бактериофага P. aeruginosa phi297 позволили предположить, что он обладает высокой литической активностью. Был выделен вирулентный мутант фага phi297vir, способный лизировать бактерии, лизогенные по фагу дикого типа. Сравнение литической активности phi297vir с литической активностью фагов из коммерческих смесей двух производителей (предприятий Нижнего Новгорода и Перми) показало, что phi297vir способен лизировать мутанты бактерий РАО1, устойчивые к фагам из коммерческих препаратов, но спектр литической активности phi297vir уже, чем у фагов коммерческих препаратов. Проведенное исследование показало, что применение неревертирующих вирулентных мутантов некоторых умеренных бактериофагов может быть целесообразно в лечении инфекций, вызванных P. aeruginosa.

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 phi297 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 of lysing the wild-type phage bacteria, 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 lytic 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.

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 vir 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.

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 lysing 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.

В настоящей работе впервые получено окончательное доказательство возможности перехода бактерий P. aeruginosa в псевдолизогенное состояние после инфекции phiKZ-подобными фагами. Показано, что решающим фактором в этом процессе является множественное заражение бактерий бактериофагами этого рода. В ходе работы были обнаружены стабильные клинические изоляты бактерий, выделяющие новые бактериофаги этого рода (Che2/2 и Che21/5), отнесенные по фенотипическим признакам и результатам анализа ДНК к видам phiKZ и EL, соответственно. Для трех бактериофагов вида EL – EL, RU и Che21/5 удалось выделить мутанты с нарушенной способностью к псевдолизогенизации. Один из мутантов фага EL имеет свойства вирулентных мутантов типичных умеренных фагов (“vir” – мутант). Он не образует псевдолизогенов и, более того, проявляет эффект доминантности при коинфекции бактерий с фагом дикого типа EL и не проявляет его при совместной инфекции бактерий с фагами дикого типа видов phiKZ и Lin68. Предполагается, что эффект псевдолизогении может быть связан с функционированием генов фагов phiKZ и EL, контролирующих продукты, сходные с репрессорами других фагов. Учитывая обнаруженное нами ранее присутствие в коммерческих фаготерапевтических препаратах phiKZ-подобных фагов дикого типа, что может создавать определенные проблемы, представляется целесообразным использовать вместо фагов дикого типа этого рода их вирулентные мутанты.

In this work, a final piece of evidence proving that bacteria Pseudomonas aeruginosa are capable of transition to the pseudolysogenic state after infection with φKZ-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 φKZ 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 φKZ and Lin68. It is assumed that the effect of pseudolysogeny may be connected with functioning of φKZ and EL genes that control the products similar to repressors of other phages. Because earlier wild-type φKZ-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.

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 bacterio phages) 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 phag e 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.

Comparison of Pseudomonas putida group of phages attributed to five species (af, ϕ15, ϕ27, ϕ2F, 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 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.

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.

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 morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ϕKZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ϕKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ϕKZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ϕKZ-like phages in respect to 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 ϕKZ-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 ϕKZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ϕKZ composition. The results of the study are discussed in terms of the evolution of these phages.

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage φKZ 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 φKZ 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 phages φKZ, Lin68, and EL, respectively) and two new genera (φKZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the φKZ species (φKZ, 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 φKZ genus. The origin and evolution of the φKZ-like phages are discussed. Analysis of protein sequences encoded by the phage φKZ genome made it possible to assume wide migration of the φKZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage φKZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.