Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.     

Morphology of Bacteriophages of Lactobacillus bulgaricus, L. lactis and L. helveticus

Eleven virulent phages isolated from cheese or yoghurt factories and active on thermophilic lactobacilli used as starters were examined by electron microscopy. Five phages active on Lactobacillus bulgaricus belonged to Bradley's group B and can be divided into two groups with different host specificity. The three phages of the first group (two isolated in France, one in the USA) differ in size; the heads are either icosahedrons or octahedrons and the tails end in short-pronged base plates. The two phages of the second group, isolated in the USA. appear very similar. These are similar in length, have collars and octahedral heads. The non-contractile tails end in clusters of short fibres. An L. lactis phage, isolated in Finland, belongs to the same morphological group. It is similar in overall appearance to the two latter phages of the second group of L. bulgaricus , and there were numerous ghosts with polytails. Five phages. active on L. helveticus belong to Bradley's group A and may be divided into two groups with different host specificity. The first group contains a phage isolated in Finland. The second group is composed of four similar phages isolated in France. The Finnish phage is the largest, but the five phages show similar morphology. They have octahedral heads, firmly attached to the tails by connector devices, and they possess necks. The contractile sheaths have a helicoidal arrangement of hollow tubular subunits. They appear contracted either in a distal or a cervical position, revealing axial cores. Short tail fibres are probably present at the tail tip.     

Morphology of the Bacteriophages of Lactic Streptococci

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.     

Escherichia coli Capsule Bacteriophages II. Morphology

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli, all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.     

Properties of Corynebacterium acnes Bacteriophage and Description of an Interference Phenomenon

Nine virulent bacteriophages of the anaerobe Corynebacterium (Propionibacterium) acnes, the P-a series, are DNA phages, with long, curved nonretractile tails (130 nm) without tail plates or fibers. They have isometric heads (420 by 460 nm), and are placed in Bradley's group B-1. There is permanent plaque suppression at highest phage concentrations. After 100- to 1,000-fold dilution, plaques are evident. The latent period is 1 h and burst size 25. Cross-neutralization data of antisera for the nine phages are similar. There is an unexplained precipitous drop in plaque-forming units during the first 5 min of neutralization, after which the rate is linear for 2 h. They are sensitive to pH extremes but are partially protected even at pH 4 or 9 by storage at 4 C. They are resistant to ether and chloroform and are inactivated within 10 min at 70 C.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The fine structure of phage HM 2 (group I) active on Clostridium saccharoperbutylacetonicum was studied by an electron microscopy with a negative-staining technique, and compared with those of more conventional types, phages HM 3 (group II) and HM 7 (group III), whose tails were clearly observed by a shadow-casting technique. This study revealed that phage HM 2 had an intricate tail which was not observed by a shadow-casting technique.

Phage HM 2 has an icosahedral head about 450 Å in diameter and a non-contractile tail about 300 Å long. The distal 130 Å of the tail axis has a width of 80 Å which is wider than the upper portion of the tail (50 to 60 Å). The distal enlargement is not seen in the hollow tail. Twelve fibrous-shaped appendages are attached symmetrically at the upper portion of tail axis and extend toward the distal base of the tail. Their length is a little shorter than 300 Å. They combine with divalent cations in the phage dilution medium, and also adsorb the host cell debris.

Phage HM 3 has an icosahedral head about 770 Å in diameter and a tail about 1000 Å long and 150 Å wide with contractile sheath. Phage HM 7 has an icosahedral head about 750 Å in diameter and a long non-contractile tail about 2000 Å long and about 120 Å wide with forked tip.

The structure of the tail of phage HM 2 is quite different from those of phages HM 3 and HM 7 hitherto described and those of the various phages of other bacteria.     

Morphology of Pasteurella multocida bacteriophages

Twenty-one tailed phages with icosahedral heads belong to the Myoviridae, Siphoviridae, and Podoviridae families and to four morphological types. Type AU, with 10 phages, has a contractile tail and is morphologically identical with coliphage P2. Lysates contain contracted tail sheaths assembled end-to-end and abnormal structures with long tails and multiple tail sheaths. Types C-2 and 32, with one and three phages, respectively, have long, noncontractile tails. Type 22 includes seven phages, has a short tail, and resembles coliphage T7. Our results agree with previous biological data and suggest that types AU, C-2, 32, and 22 correspond to four different phage species.     

Structure of the receptor-binding protein of bacteriophage det7: a podoviral tail spike in a myovirus

A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The morphological properties of the twelve previously described HM-phages were examined by electron microscopy. Specimens were prepared by air-drying and shadow-casting method using purified phage suspensions. As a result, the HM-phages were classified into three morphologically distinct groups, 1, 11 and 111. Group 1 phages were HM 1, HM 2, HM 8, HM 9, HM 10, HM 11 and HM 12. These phages had a spherical head about 100 mμ in diameter and a rudimentary tail. Group 11 phages were HM 3, HM 4, HM 5 and HM 6. These phages had a spherical head about 100 mμ in diameter and a tail with contractile sheath, and the normal tail of these phages was about 100 mμ in length, and the contracted sheath was about 50 mμ in length, Group 111 phage was HM 7 alone. This phage had a spherical head about 120 mμ in diameter and a relatively long tail about 350 mμ in length.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.     

A touch of glue to complete bacteriophage assembly: the tail‐to‐head joining protein (THJP) family

Bacteriophage SPP1 is a nanomachine built to infect the bacterium Bacillus subtilis. The phage particle is composed of an icosahedric capsid, which contains the viral DNA, and a long non‐contractile tail. Capsids and tails are produced in infected cells by two distinct morphogenetic pathways. Characterization of the suppressor‐sensitive mutant SPP1sus82 showed that it produces DNA‐filled capsids and tails but is unable to assemble complete virions. Its purified tails have a normal length but lack a narrow ring that tapers the tail end found at the tail‐to‐head interface. The mutant is defective in production of gp17. The gp17 ring is exposed in free tails competent for viral assembly but becomes shielded in the final virion structure. Recombinant gp17 is active in an in vitro assay to stick together capsids and tails present in extracts of SPP1sus82‐infected cells, leading to formation of infectious particles. Gp17 thus plays a fundamental role in the tail‐to‐head joining reaction, the ultimate step of virus particle assembly. This is the conserved function of gp17 and its structurally related proteins like lambda gpU. This family of proteins can also provide fidelity to termination of the tail tube elongation reaction in a subset of phages including coliphage lambda.     

Characterization of Vibrio cholerae Bacteriophages Isolated from the Environmental Waters of the Lake Victoria Region of Kenya

Over the last decade, cholera outbreaks have become common in some parts of Kenya. The most recent cholera outbreak occurred in Coastal and Lake Victoria region during January 2009 and May 2010, where a total of 11,769 cases and 274 deaths were reported by the Ministry of Public Health and Sanitation. The objective of this study is to isolate Vibrio cholerae bacteriophages from the environmental waters of the Lake Victoria region of Kenya with potential for use as a biocontrol for cholera outbreaks. Water samples from wells, ponds, sewage effluent, boreholes, rivers, and lakes of the Lake Victoria region of Kenya were enriched for 48 h at 37 °C in broth containing a an environmental strain of V. cholerae. Bacteriophages were isolated from 5 out of the 42 environmental water samples taken. Isolated phages produced tiny, round, and clear plaques suggesting that these phages were lytic to V. cholerae. Transmission electron microscope examination revealed that all the nine phages belonged to the family Myoviridae, with typical icosahedral heads, long contractile tails, and fibers. Head had an average diameter of 88.3 nm and tail of length and width 84.9 and 16.1 nm, respectively. Vibriophages isolated from the Lake Victoria region of Kenya have been characterized and the isolated phages may have a potential to be used as antibacterial agents to control pathogenic V. cholerae bacteria in water reservoirs.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Conserved translational frameshift in dsDNA bacteriophage tail assembly genes

A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.     

Electron-microscopic study of the serological affinity between the antigenic components of phages T4 and DDVI.

In an investigation of the antigenic fine structure of phages T4 and DDVI with the use of the neutralization reaction and electron-microscopic observation of the phage-antibody complexes, it has been possible to establish that the head of phage T4 consists of proteins which have antigenic determinants of two types: The first type is identical to the antigens of the head of phage DDVI, and the second type is apparently absent in phage DDVI. The phage DDVI head contains mostly determinants which are common to the phage T4 head, since it was not possible to detect antigenically specific components in the phage DDVI head. The tail sheaths of phage T4 and DDVI appear to be identical in the antigenic respect. A difference has been observed in the fibers and the base plates of the phages investigated. The presence of the following three types of antigens has been established: 1) common to phages T2, T4, and DDVI, 2) common to phages T4 and DDVI, and 3) specific for each phage investigated.     

Degrees of relatedness of T-even type E. coli phages using different or the same receptors and topology of serologically cross-reacting sites

The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated. Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein. Absence of cross-reactivity appears to reflect high degrees of dissimilarity. A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber. Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages. Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk “Viili”

“Viili,” a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Classification of virulent and temperate bacteriophages of Listeria spp. on the basis of morphology and protein analysis.

A set of 22 phages of Listeria species (listeriaphages) (21 temperate and 1 virulent) were compared on the basis of morphology and protein composition. All 22 phages had icosahedral heads and exhibited either contractile or noncontractile tails. They represented two different morphotypes. Twenty phages belonged to the Siphoviridae family and could be differentiated only on the basis of tail length. Accordingly, they could be assigned to previously defined listeriaphage species. Two other phages were classified as members of the Myoviridae family, one of which (A511) should be regarded as a new species. It was found to be substantially different from all other known listeriaphages. All phages exhibited typical protein profiles, which were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent laser densitometrical analysis of the gels. It was then possible to distinguish eight protein subgroups on the basis of unique protein patterns. This classification corresponds well to the previous groupings based on host range. Most of the phages revealed two or three major proteins ranging from 21 to 24 kDa and 30 to 36 kDa. In addition, at least 10 minor proteins could be observed for each phage. Our results indicate that the major proteins are structural proteins of the capsid and tail, and the protein band ranging from 30 to 35 kDa could clearly be assigned to the proteins of the phage capsid.     

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 C2, C5, and CD119. 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.     

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.