Characterization of DC1, a broad-host-range Bcep22-like podovirus

Bcep22-like phages are a recently described group of podoviruses that infect strains of Burkholderia cenocepacia. We have isolated and characterized a novel member of this group named DC1. This podovirus shows many genomic similarities to BcepIL02 and Bcep22, but it infects strains belonging to multiple Burkholderia cepacia complex (BCC) species.     

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.     

A Virulent Phage Infecting Lactococcus garvieae,with Homology to Lactococcus lactis Phages

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Erratum: Causes and Consequences of Bacteriophage Diversification via Genetic Exchanges across Lifestyles and Bacterial Taxa

Bacteriophages (phages) evolve rapidly by acquiring genes from other phages. This results in mosaic genomes. Here, we identify numerous genetic transfers between distantly related phages and aim at understanding their frequency, consequences, and the conditions favoring them. Gene flow tends to occur between phages that are enriched for recombinases, transposases, and nonhomologous end joining, suggesting that both homologous and illegitimate recombination contribute to gene flow. Phage family and host phyla are strong barriers to gene exchange, but phage lifestyle is not. Even if we observe four times more recent transfers between temperate phages than between other pairs, there is extensive gene flow between temperate and virulent phages, and between the latter. These predominantly involve virulent phages with large genomes previously classed as low gene flux, and lead to the preferential transfer of genes encoding functions involved in cell energetics, nucleotide metabolism, DNA packaging and injection, and virion assembly. Such exchanges may contribute to the observed twice larger genomes of virulent phages. We used genetic transfers, which occur upon coinfection of a host, to compare phage host range. We found that virulent phages have broader host ranges and can mediate genetic exchanges between narrow host range temperate phages infecting distant bacterial hosts, thus contributing to gene flow between virulent phages, as well as between temperate phages. This gene flow drastically expands the gene repertoires available for phage and bacterial evolution, including the transfer of functional innovations across taxa.     

Genomic characterization of two Staphylococcus epidermidis bacteriophages with anti-biofilm potential

ABSTRACT: BACKGROUND: Staphylococcus epidermidis is a commensal bacterium but can colonize the hospital environment due to its ability to form biofilms favouring adhesion to host tissues, medical devices and increasing resistance to antibiotics. In this context, the use of phages to destroy biofilms is an interesting alternative. RESULTS: The complete genomes of two Staphylococcus epidermidis bacteriophages, vB_SepiSphiIPLA5 and vB_SepiS-phiIPLA7, have been analyzed. Their genomes are 43,581 bp and 42,123 bp, and contain 67 and 59 orfs. Bioinformatic analyses enabled the assignment of putative functions to 36 and 29 gene products, respectively, including DNA packaging and morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, regulation, modification and replication. A point mutation in vB_SepiS-phiIPLA5 lysogeny control-associated genes explained its strictly lytic behaviour. Comparative analysis of phi- IPLA5 and phi-IPLA7 genome structure resembled those of S. epidermidis phiPH15 and phiCNPH82 phages. A mosaic structure of S. epidermidis prophage genomes was revealed by PCR analysis of three marker genes (integrase, major head protein and holin). Using these genes, high prevalence (73%) of phage DNA in a representative S. epidermidis strain collection consisting of 60 isolates from women with mastitis and healthy women was determined. Putative pectin lyase-like domains detected in virion-associated proteins of both phages could be involved in exopolysaccharide (EPS) depolymerization, as evidenced by both the presence of a clear halo surrounding the phage lysis zone and the phage-mediated biofilm degradation. CONCLUSIONS: Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have a mosaic structure similar to other widespread S. epidermidis prophages. Virions of these phages are provided of pectin lyase-like domains, which may be regarded as promising anti-biofilm tools.     

Characterization and genomic analysis of phage asccphi28, a phage of the family Podoviridae infecting Lactococcus lactis

Bacteriophage asccphi28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccphi28 and its full genome sequence. Phage asccphi28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccphi28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccphi28 was found to be 121 +/- 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccphi28 more closely resembles streptococcal phage Cp-1 and the phi29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.     

Exclusion of glucosyl-hydroxymethylcytosine DNA containing bacteriophages is overcome by the injected protein inhibitor IPI*

The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1(-) phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1(-) phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages.     

Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.     

Phylogenetic analyses suggest lateral gene transfer from the mitochondrion to the apicoplast

The genomes of the three temperate bacteriophages contained in the chromosome of Staphylococcus aureus 8325 have been extracted from the sequence database and analyzed. phi 11, phi 12 and phi 13 are members of the same lytic group but different serogroups and consequently co-habitate the same host cell. Their genomes are approximately 42 kb to 45 kb and contain about 90 ORFs of at least 50 codons. Of these, about 50 have similarities to known genes or to genes of other staphylococcal phages. Each of the phages clusters within a homology group that share large regions of sequence identity while intergroup homology is comparatively low. The arrangement of genes on the chromosomes of the three phages is similar and consistent with current modular theory of phage gene organization. The replicated genomes appear to be packaged by different mechanisms. Phage phi 11 and phi 12 have been found to contain sequences consistent with pac-site phages while phi 13 has sequences consistent with cos-site phages. The attBsite for phi 11 is located in an intergenic region of the S. aureus chromosome while phi 12 and phi 13 integrate into specific genes. The phi 12 att-site is within an unknown gene, but the phi 13 att-site is within the beta-toxin gene. In contrast to the other two phages, phi 13 also introduces the staphylokinase gene (sak) and a second gene related to expression of fib.     

Widespread distribution of a group I intron and its three deletion derivatives in the lysin gene of Streptococcus thermophilus bacteriophages

Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilus bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillus phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from Lactococcus, Lactobacillus, and Bacillus phages.     

Horizontal gene transfer and the evolution of microvirid coliphage genomes

Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family Microviridae, including phiX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the Microviridae and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages.     

Isolation and characterization of a novel Enterococcus faecalis bacteriophage φEF24C as a therapeutic candidate

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage φEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of φEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that φEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that φEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c . 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that φEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, φEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.     

Transposition of lambda placMu is mediated by the A protein altered at its carboxy-terminal end

Lambda placMu phages are derivatives of bacteriophage lambda that use the transposition machinery of phage Mu to insert into chromosomal and cloned genes. When inserted in the proper fashion, these phages yield stable fusions to the Escherichia coli lac operon in a single step. We have determined the amount of DNA from the c end of phage Mu present in one of these phages, lambda placMu3, and have shown that this phage carries a 3137-bp fragment of Mu DNA. This DNA segment carries the Mu c-end attachment site and encodes the Mu genes cts62, ner+, and gene A lacking 179 bp at its 3' end (A'). The product of this truncated gene A' retains transposase activity and is sufficient for the transposition of lambda placMu. This was demonstrated by showing that lambda placMu derivatives carrying the A am1093 mutation in the A' gene are unable to transpose by themselves in a Su- strain, but their transposition can be triggered by coinfection with lambda pMu507(A+ B+). We have constructed several new lambda placMu phages that carry the A' am1093 gene and the kan gene, which confers resistance to kanamycin. Chromosomal insertions of these new phages are even more stable than those of the previously reported lambda placMu phages, which makes them useful tools for genetic analysis.     

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. 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.     

Two Phages,phiIPLA-RODI and phiIPLA-C1C,Lyse Mono- and Dual-Species Staphylococcal Biofilms

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10⁻⁷ ± 2.34 × 10⁻⁹ and 1.1 × 10⁻⁷ ± 2.08 × 10⁻⁹, respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.