Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

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.     

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.     

Taxonomic differentiation of 101 lactococcal bacteriophages and characterization of bacteriophages with unusually large genomes

Sixty-three virulent bacteriophages of Lactococcus lactis were differentiated by DNA-DNA hybridization. The results, including those of a previous classification of 38 phages of the same bacterial species (P. Relano, M. Mata, M. Bonneau, and P. Ritzenthaler, J. Gen. Microbiol. 133:3053-3063, 1987) show that 48% of the phages analyzed belong to a unique DNA homology group (group III). Phages of this most abundant group had small isometric heads. Group I comprised 29% of the phages analyzed and was characterized by a small phage genome (19 to 22 kilobases) and a particular morphology with a prolate head. Like group III, this group contained representative phages of other classifications. Group II (21%) included virulent and temperate phages with small isometric heads. Two large isometric-headed phages, phi 109 and phi 111, were not related to the three DNA homology groups I, II, and III. The genome of phi 111 was unusually large (134 kilobases) and revealed partial DNA homology with another large isometric phage, 1289, described by Jarvis (type e) (A. W. Jarvis, Appl. Environ. Microbiol. 47:343-349, 1984). The protein compositions of phi 111 and 1289 were similar (three common major proteins of 21, 28, and 32 kilodaltons).     

Some Bacteriophages Active Against Rhizobium trifolii Strain W19

Fourteen soil bacteriophages active against Rhizobium trifolii W19 have been studied which fall into four structural groups. Group 1 phages have contractile tails. Some particles show double base plates to which at least three spikes are attached, and fibers are attached to the base plates. Group 2 phages also have contractile tails. At least five spikes are attached to the base plate, and there are spherical bodies adjacent to the tail, at the ends of fibers attached to the tail base. Group 3 phages have contractile tails, but are larger than phages of groups 1 and 2. The end of the tail has a complex structure. Group 4 phages have long, noncontractile tails.     

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.     

Temperate phages of Clostridium perfringens type C1.

Four phages isolated from carrier strains of Clostridium perfringens type C belong to two classes. The three phages of class I, c1, c3, and c4, and homoimmune and serologically closely related. The phage of class II, c5, is heteroimmune to the class I phages and not related to them serologically. Transduction experiments with several of the phages were negative. Mutants of the indicator strain with surface alterations occurred spontaneously in stock cultures. Electron micrographs show the phages of each class to be distinct yet similar, having polyhedral heads of about the same diameter 55 nm, and long, flexible tails without sheaths or collars. Phages c4 and c5 were characterized for their lysogenic properties. Phage c4 was inducible with mitomycin C. Both c4 and c5 were temperate viruses by the test of stability of their respective lysogens to phage-specific antisera.     

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.     

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.     

The isolation and characterization of bacteriophages infecting obligately thermophilic strains of Bacillus

Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50 degrees C for 4-5 h but at 70 degrees C the plaque-forming units decreased by between 10(2)- and 10(7)-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that 'B. caldotenax' has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of 'B. caldotenax' and 'B. caldovelox' by phage JS017 has been observed.     

Genetic studies of lactococcal bacteriophages—taxonomic differentiations and DNA analysis: evidence for 3' cohesive ends

Twenty-four bacteriophages of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were classified. Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies. These criteria showed that prolate phages are quite homogeneous. In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions. Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52. These two families were clearly defined. The unique phage which does not fit in either group probably belongs to a third one which is much less represented. No obvious relationships between these criteria and the lytic spectra were detected. Evidence of the presence of cohesive ends in phage genomes is also presented in this study. A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.     

Structural aberrations in group A Staphylococcus bacteriophages.

Six related Staphylococcus phages spontaneously produced various abnormal head and tail structures: (i) giant capsids which were tailed and apparently contained nucleic acid; (ii) regular and irregular smooth polyheads; (iii) heads and polyheads with wavy outlines; (iv) mottled heads and polyheads; (v) abnormally long and short tails; and (vi) "double capsids" connected by a small bridge. Some of these structures are rare, or have not yet been reported. The frequency os specific aberrant particles varied from one phage to another. Length distribution of smooth irregular polyheads and of abnormal tails indicated that these structures assemble at random from protein synthesized in excess. These phages represent an interesting model for genetic and morphogentic studies.     

The morphology and nucleotide composition of DNA of Citrobacter phages

Citrobacter phages 38/37, 31/37, 40/1 and 8/5, isolated from lysogenic cultures, were concentrated and purified by 2 cycles of differential centrifugation. Electron microscopy of the phages has shown that their particles have similar morphology and that they relate to the morphological group A1. The heads of the phages are hexagonal, 50 +/- 2 nm in diameter. The tail of the phage is straight, 112-152 nm in length, with a contracting sheath 11.5-12.5 nm wide. The tails of the phages 38/37 and 40/1 were found to be slightly longer in comparison with the phages 31/37 and 8/5. Chromatographic investigation of DNA preparations of the phages revealed the presence of 4 nitrous bases. Identification of the latter permitted us to relate them to common nitrous bases. DNA of the phages is double-stranded and belongs to a weakly expressed guanine-cytosine type. The content of guanine and cytosine in DNA of the phage 38/37 amounts to 56.68%, that of the phage 31/37 to 56.75, of the phage 40/1 to 57.36% and of the phage 8/5 to 55.58%. No substantial variations were observed in the DNA composition of the phages.     

Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.     

Unusual Bacteriophages in Salmonella newport

Six phages were isolated from sewage and from a lysogenic strain. Three of them, belonging to a new morphological group, had contractile tails and elongated heads with axial ratios of 2.4:1. Two phages, possessing short tails and very long heads with axial ratios of 3.5:1, were new isolates of an extremely rare group. Phages of both groups formed polyheads of various sizes and shapes. The last phage, 61 nm in diameter, seemed to have a tail-like appendage. All phages had double-stranded DNA, were active on enterobacteria only, and differed in their host range. The first five phages seemed to be Salmonella specific.     

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.     

Morphology and General Characteristics of Bacteriophages Infectious to Robinia pseudoacacia Mesorhizobia

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley’s classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10–25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100–120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from ~39 kb for group C phages to ~80 kb for B2.     

Preliminary Characterization of Bacteriophages Infecting the Thermophilic Actinomycete Thermomonospora

Bacteriophages lysing strains of Thermomonospora alba and T. fusca were isolated, following specific enrichment, from vegetable composts. Four Thermomonospora phages were distinguished by plaque morphology and host range. Electron microscopy of phage particles, termperature inactivation profiles, and electrophoretic analyses of major virion proteins and genomic DNA were used in the comparison and initial characterization of these phages. The four phages studied possessed polyhedral heads and long tails; genomes were linear double-stranded DNA molecules, 35 to 45 kilobases in length, which probably contain cohesive ends. Transfection of Thermomonospora protoplasts with purified genomic DNA from one of the phages was demonstrated.     

Lysogenic strains of lactic Acid streptococci and lytic spectra of their temperate bacteriophages

A total of 113 strains of mesophilic strains lactic streptococci of the species Streptococcus lactis, S. lactis subsp. diacetilactis, and S. cremoris, chosen from 291 strains that had been previously classified into six groups on the basis of their sensitivity to 132 virulent phages, were subjected to induction with mitomycin C. Among these strains, 43% produced phages capable of forming plaques of lysis on an indicator strain either spontaneously or after induction. There was a close correlation between the lytic spectra of temperate and virulent phages. Among the strains studied, 25% were shown to be indicator strains. These results emphasized the high probability of development of temperate phages in a starter culture containing mesophilic lactic streptococci and therefore their importance as a cause of accidents in cheese making.     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.