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.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of the ϕKZ-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 morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ?KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ?KZ 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 ?KZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ?KZ-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 ?KZ-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 ?KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ?KZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Evolutionary history of bacteriophages with double-stranded DNA genomes

Background

Reconstruction of evolutionary history of bacteriophages is a difficult problem because of fast sequence drift and lack of omnipresent genes in phage genomes. Moreover, losses and recombinational exchanges of genes are so pervasive in phages that the plausibility of phylogenetic inference in phage kingdom has been questioned.

Results

We compiled the profiles of presence and absence of 803 orthologous genes in 158 completely sequenced phages with double-stranded DNA genomes and used these gene content vectors to infer the evolutionary history of phages. There were 18 well-supported clades, mostly corresponding to accepted genera, but in some cases appearing to define new taxonomic groups. Conflicts between this phylogeny and trees constructed from sequence alignments of phage proteins were exploited to infer 294 specific acts of intergenome gene transfer.

Conclusion

A notoriously reticulate evolutionary history of fast-evolving phages can be reconstructed in considerable detail by quantitative comparative genomics.

Open peer review

This article was reviewed by Eugene Koonin, Nicholas Galtier and Martijn Huynen.     

Classification of virulent bacteriophages of Streptococcus salivarius subsp. thermophilus isolated from yoghurt and Swiss-type cheese

Summary Twelve isometric-headed bacteriophages virulent against Streptococcus salivarius subsp. thermophilus were differentiated into three subgroups by analysis of the phage genomes and the structural proteins. Subgroup I is composed of two phages (P6 and P8) with a genome size of 41.2 and 44.2 kb pairs, respectively, complete DNA homology, and identical protein composition (main proteins of sizes 39.8, 24.0, 14.8 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis). One phage (a10/J9) with low DNA homology to the other phages was classified into subgroup II. Subgroup III consists of nine phages with a genome size of 33.8 to 36.7 kb pairs and two major structural proteins (30.9 and 24.0 kilodaltons, or 30.9 and 26.3 kilodaltons). In general, phages with different host spectra revealed different restriction enzyme patterns, and DNA homologies of various degrees were detected among all phages tested.     

Ecology of bacteriophages infecting activated sludge bacteria.

Little is known about the endemic bacteriophages of activated sludge. In this investigation 49 virus-host systems were studied by isolating co-occurring bacteria and bacteriophages from the aeration basin of a sewage treatment plant during 5 successive weeks. The phage titers were high and fluctuated during the time period. The occurrence of phage-sensitive and -resistant hosts did not depend on the presence or absence of phages. Several phage-host systems expressed variable plating efficiencies. In addition, phages with broad host ranges were observed. These results show that phages are an active part of this ecosystem and that they may exert selection pressure for phage resistance on their bacterial host populations.     

Ecology of bacteriophages infecting activated sludge bacteria.

Little is known about the endemic bacteriophages of activated sludge. In this investigation 49 virus-host systems were studied by isolating co-occurring bacteria and bacteriophages from the aeration basin of a sewage treatment plant during 5 successive weeks. The phage titers were high and fluctuated during the time period. The occurrence of phage-sensitive and -resistant hosts did not depend on the presence or absence of phages. Several phage-host systems expressed variable plating efficiencies. In addition, phages with broad host ranges were observed. These results show that phages are an active part of this ecosystem and that they may exert selection pressure for phage resistance on their bacterial host populations.     

Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibres

Molecular analysis reveals a surprising sharing of short gene segments among a variety of large double-stranded DNA bacteriophages of enteric bacteria. Ancestral genomes from otherwise unrelated phages, including λ Mu, P1, P2 and T4, must have exchanged parts of their tail-fibre genes, Individual genes appear as mosaics with parts derived from a common gene pool. Therefore, horizontal gene transfer emerges as a major factor in the evolution of a specific part of phage genomes. Current concepts of homologous recombination cannot account for the formation of such chimeric genes and the recombinational mechanisms responsible are not known. However, recombination sites for DNA invertases and recombination site-like sequences are present at the boundaries of gene segments conferring the specificity for the host receptor. This, together with the properties of the DNA inversion mechanism, suggests that these site-specific recombination enzymes could be responsible for the exchange of host-range determinants.     

Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages.

In complex DNA bacteriophages like lambda, T4, T7, P22, P2, the DNA is packaged into a preformed precursor particle which sometimes has a smaller size and often a shape different from that of the phage head. This packaging mechanism is different from the one suggested for the RNA phages, according to which RNA nucleates the shell formation. The different mechanisms could be understood by comparing the genomes to be packaged: single stranded fII RNA has a very compact structure with high helix content. It might easily form quasispherical structures in solution (as seen in the electron microscope by Thach & Thach (1973)) around which the capsid could assemble. Double stranded phage DNA, on the other hand, is a rigid molecule which occupies a large volume in solution and has to be concentrated 15-fold during packaging into the preformed capsid, and the change in the capsid structure observed hereby might provide the necessary DNA condensation energy.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of 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.     

Pseudolysogeny of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> bacteria infected with φKZ-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 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.     

A diversity of bacteriophage forms and genomes can be isolated from the surface sands of the Sahara Desert

The surface sands of the Sahara Desert are exposed to extremes of ultraviolet light irradiation, desiccation and temperature variation. Nonetheless, the presence of bacteria has recently been demonstrated in this environment by cultivation methods and by 16S rDNA analyses from total DNA isolated from surface sands. To discern the presence of bacteriophages in this harsh environment, we searched for extracellular phages and intracellularly located phages present as prophages or within pseudolysogens. Mild sonication of the sand, in different liquid culture media, incubated with and without Mitomycin-C, was followed by differential centrifugation to enrich for dsDNA phages. The resulting preparations, examined by electron microscopy, revealed the presence of virus-like particles with a diversity of morphotypes representative of all three major double-stranded DNA bacteriophage families (Myoviridae, Siphoviridae and Podoviridae). Moreover, pulsed-field gel electrophoresis of DNA, extracted from the enriched bacteriophage preparations, revealed the presence of distinct bands suggesting the presence of putative dsDNA phage genomes ranging in size from 45 kb to 270 kb. Characterization of the bacteriophages present in the surface sands of the Sahara Desert extends the range of environments from which bacteriophages can be isolated, and provides an important point of departure for the study of phages in extreme terrestrial environments.Magali Prigent and Magali Leroy contributed equally to this work     

Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.     

Pseudomonas aeruginosa bacteriophages with DNA structure similar to the DNA structure of Mu1 phage. II. Evidence for similarity between D3112, B3, and B39 bacteriophages: analysis of DNA splits by restriction endonucleases, isolation of D3112 and B3 recombinant phages

It is found that bacteriophages B3 and B39 specific for Pseudomonas aeruginosa have the same genome structure as previously described phage D3112. On the right (S) end of their genomes a variable non-phage DNA is located (approximately 0.9-2.5 kilobases for different phages). It is probable that this variable DNa has its origin from different regions of bacterial chromosome. In genome of one of the phages, B3 phage, such variable DNA (not more than 150 base pairs) was found on the left end of DNA molecule. Isolation of a viable B3XD3112 recombinant phage and analysis of its genome with restriction technique and with studies of homo- and heteroduplex molecules had confirmed genetical relationship of B3 and D3112. Some essential non-homology of B3 and D3112 DNAs have been found on the right ends of genomes of the phages.     

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

High diversity and novel species of Pseudomonas aeruginosa bacteriophages

The diversity of Pseudomonas aeruginosa bacteriophages was investigated using a collection of 68 phages isolated from Central Mexico. Most of the phages carried double-stranded DNA (dsDNA) genomes and were classified into 12 species. Comparison of the genomes of selected archetypal phages with extant sequences in GenBank resulted in the identification of six novel species. This finding increased the group diversity by ~30%. The great diversity of phage species could be related to the ubiquitous nature of P. aeruginosa.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages

Background

Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.

Results

P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.

Conclusions

This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users.