Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C 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 phiEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phiEF24C 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 phiEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phiEF24C 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 phiEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phiEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.     

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.     

Complete genomic sequence of bacteriophage phiEcoM-GJ1, a novel phage that has myovirus morphology and a podovirus-like RNA polymerase

The complete genome of phiEcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli sigma(70)-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that phiEcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.     

The terminally redundant, nonpermuted genome of Listeria bacteriophage A511: a model for the SPO1-like myoviruses of gram-positive bacteria

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (approximately 6 kb) and K (approximately 20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.     

Morphological,host range,and genetic characterization of two coliphages

Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food.     

Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.     

Three Prochlorococcus Cyanophage Genomes: Signature Features and Ecological Interpretations

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.     

Three Prochlorococcus Cyanophage Genomes: Signature Features and Ecological Interpretations

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.     

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.     

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.     

An examination of the bacteriophages and bacteria of the Namib desert

Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth's terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55 approximately 65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94 approximately 98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and 8-Proteobacteria groups were also observed.     

Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.     

Genome of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2

Despite the fact that multidrug-resistant Klebsiella sp. strains emerge rapidly (Xu J, et al., Adv. Mater. Res. 268-270:1954-1956, 2011) and bacteriophages have been reported to be useful in controlling these bacteria (Kumari S, Harjai K, Chhibber S, J. Med. Microbiol. 60:205-210, 2011), the complete genome sequences of only five Klebsiella phages (four siphoviruses and one myovirus) can be found in databases. In this paper, we report on the complete genome sequence of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2. With a genome size of 345,809 bp, this is the second largest myovirus and the largest Klebsiella phage sequenced to date. This phage differs substantially from other myoviruses since 411 out of 534 vB_KleM_RaK2 open reading frames have no known functions and lack any reliable database matches. Comparative analysis of the genome sequence of vB_KleM_RaK2 suggests that this phage forms a distinct phylogenetic branch within the family Myoviridae of tailed bacteriophages.     

Morphology of Brochothrix thermosphacta phages

Twenty-one Brochothrix thermosphacta phages were examined by electron microscopy. They had isometric heads and contractile or long and noncontractile tails, and were grouped into three species belonging to the Myoviridae (species A19) or Siphoviridae (species NF5 and BL3) families of tailed phages. Species A19 has highly characteristic morphological features and resembles well-known phages of Bacillus, Lactobacillus, Staphylococcus and Streptococcus, indicating phylogenetic relationships between these phages as well as their hosts.     

Genomic analysis of oceanic cyanobacterial myoviruses compared with T4‐like myoviruses from diverse hosts and environments

T4‐like myoviruses are ubiquitous, and their genes are among the most abundant documented in ocean systems. Here we compare 26 T4‐like genomes, including 10 from non‐cyanobacterial myoviruses, and 16 from marine cyanobacterial myoviruses (cyanophages) isolated on diverse Prochlorococcus or Synechococcus hosts. A core genome of 38 virion construction and DNA replication genes was observed in all 26 genomes, with 32 and 25 additional genes shared among the non‐cyanophage and cyanophage subsets, respectively. These hierarchical cores are highly syntenic across the genomes, and sampled to saturation. The 25 cyanophage core genes include six previously described genes with putative functions (psbA, mazG, phoH, hsp20, hli03, cobS), a hypothetical protein with a potential phytanoyl‐CoA dioxygenase domain, two virion structural genes, and 16 hypothetical genes. Beyond previously described cyanophage‐encoded photosynthesis and phosphate stress genes, we observed core genes that may play a role in nitrogen metabolism during infection through modulation of 2‐oxoglutarate. Patterns among non‐core genes that may drive niche diversification revealed that phosphorus‐related gene content reflects source waters rather than host strain used for isolation, and that carbon metabolism genes appear associated with putative mobile elements. As well, phages isolated on Synechococcus had higher genome‐wide %G+C and often contained different gene subsets (e.g. petE, zwf, gnd, prnA, cpeT) than those isolated on Prochlorococcus. However, no clear diagnostic genes emerged to distinguish these phage groups, suggesting blurred boundaries possibly due to cross‐infection. Finally, genome‐wide comparisons of both diverse and closely related, co‐isolated genomes provide a locus‐to‐locus variability metric that will prove valuable for interpreting metagenomic data sets.     

Morphological and genetic diversity of temperate phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.     

Ecogenomics and genome landscapes of marine Pseudoalteromonas phage H105/1

Marine phages have an astounding global abundance and ecological impact. However, little knowledge is derived from phage genomes, as most of the open reading frames in their small genomes are unknown, novel proteins. To infer potential functional and ecological relevance of sequenced marine Pseudoalteromonas phage H105/1, two strategies were used. First, similarity searches were extended to include six viral and bacterial metagenomes paired with their respective environmental contextual data. This approach revealed ‘ecogenomic'' patterns of Pseudoalteromonas phage H105/1, such as its estuarine origin. Second, intrinsic genome signatures (phylogenetic, codon adaptation and tetranucleotide (tetra) frequencies) were evaluated on a resolved intra-genomic level to shed light on the evolution of phage functional modules. On the basis of differential codon adaptation of Phage H105/1 proteins to the sequenced Pseudoalteromonas spp., regions of the phage genome with the most ‘host''-adapted proteins also have the strongest bacterial tetra signature, whereas the least ‘host''-adapted proteins have the strongest phage tetra signature. Such a pattern may reflect the evolutionary history of the respective phage proteins and functional modules. Finally, analysis of the structural proteome identified seven proteins that make up the mature virion, four of which were previously unknown. This integrated approach combines both novel and classical strategies and serves as a model to elucidate ecological inferences and evolutionary relationships from phage genomes that typically abound with unknown gene content.     

Reticulate representation of evolutionary and functional relationships between phage genomes

Bacteriophage genomes show pervasive mosaicism, indicating the importance of horizontal gene exchange in their evolution. Phage genomes represent unique combinations of modules, each of them with a different phylogenetic history. The traditional classification, based on a variety of criteria such as nucleic acid type (single/double-stranded DNA/RNA), morphology, and host range, appeared inconsistent with sequence analyses. With the genomic era, an ever increasing number of sequenced phages cannot be classified, in part due to a lack of morphological information and in part to the intrinsic incapability of tree-based methods to efficiently deal with mosaicism. This problem led some virologists to call for a moratorium on the creation of additional taxa in the order Caudovirales, in order to let virologists discuss classification schemes that might better suit phage evolution. In this context, we propose a framework for a reticulate classification of phages based on gene content. Starting from gene families, we built a weighted graph, where nodes represent phages and edges represent phage-phage similarities in terms of shared genes. We then apply various measures of graph topology to analyze the resulting graph. Most double-stranded DNA phages are found in a single component. The values of the clustering coefficient and closeness distinguish temperate from virulent phages, whereas chimeric phages are characterized by a high betweenness coefficient. We apply a 2-step clustering method to this graph to generate a reticulate classification of phages: Each phage is associated with a membership vector, which quantitatively characterizes its membership to the set of clusters. Furthermore, we cluster genes based on their "phylogenetic profiles" to define "evolutionary cohesive modules." In virulent phages, evolutionary modules span several functional categories, whereas in temperate phages they correspond better to functional modules. Moreover, despite the fact that modules only cover a fraction of all phage genes, phage groups can be distinguished by their different combination of modules, serving the bases for a higher level reticulate classification. These 2 classification schemes provide an automatic and dynamic way of representing the relationships within the phage population and can be extended to include newly sequenced phage genomes, as well as other types of genetic elements.     

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.     

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.