Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins.

A comparative study of 30 phages of Streptococcus thermophilus was performed based on DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and host range data. All phages exhibited distinct DNA restriction profiles, with some phages displaying similarly sized restriction fragments. DNA homology was shown to be present among all 30 phages. The phages could be divided into two groups on the basis of their packaging mechanism as was derived from the appearance of submolar DNA fragments in restriction enzyme digests and the presence (cos-containing phages) or absence (pac-containing phages) of cohesive genomic extremities. Interestingly, the 19 identified cos-containing phages possessed two major structural proteins (32 and 26 kDa) in contrast to the remaining 11 pac-containing phages, which possessed three major structural proteins (41, 25, and 13 kDa). Southern hybridization demonstrated that all pac-containing phages tested contain homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibit homology to the gene specifying one of the structural components of the cos-containing phage phi 7201. Fifty-seven percent of the phages (both cos and pac containing) possessed the previously identified 2.2-kb EcoRI fragment of the temperate S. thermophilus phage Sfi18 (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994). No obvious correlation was detected between grouping based on packaging mechanism and host range data obtained with 39 industrial S. thermophilus strains.     

Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance

Sequence data derived from the Streptococcus thermophilus phages phiO1205 and phi7201 indicated that each of these phages contains a distinct DNA region dedicated to replication. Southern blotting experiments showed that phages infecting S. thermophilus may be divided into at least two groups, each containing the presumptive replication functions of either φO1205 (group I) or φ7201 (group II). Specific regions from the putative replication module of each of the two phages were examined for their ability to provide phage resistance.     

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

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

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like 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).     

Phi SC623, a temperate actinophage of Streptomyces coelicolor Müller, and its relatives phi SC347 and phi SC681

Three species-specific, temperate actinophages of Streptomyces coelicolor Müller, phi SC623, phi SC347 and phi SC681, were compared with respect to host range, virion structure, antiserum cross-inactivation, DNA-restriction pattern, DNA hybridization, and DNA base composition. The restriction map of phi SC623 (57 kb) was established with eight restriction enzymes; the homologies of this phage with phi SC347 and phi SC681 suggested that it might be a hybrid phage composed of approximately equal parts homologous to one of the other two phages. No homology was detected between phi SC623 and R4, a temperate, wide-host-range phage which can also lysogenize S. coelicolor Müller.     

2 types of molecular structure (composition) of a genome in one species of transposable bacteriophages of Pseudomonas aeruginosa

Comparison of heteroduplexes (HD) between DNAs of different transposable phages of Pseudomonas aeruginosa belonging to two previously described subgroups (D3112 and B3) revealed two types of structure (composition) of the bacteriophages, designated "type A" and "type B". The properties of genome structure of type A (phages of D3112 subgroup) are as follows: high level of conservation (up to 70% of genomes of different phages are represented as blocks of homologous DNA sequences); substitutions in genomes revealed as nonhomology regions in HD are, as a rule, small and located in certain sites; the distribution of the nonhomologous regions in HD of these phages is highly reproducible in independent experiments. Bacteriophages of subgroup B3 have genomes of type B: only a small part (approx. 30%) of genomes retain homology general for all of the phages; the nonhomologous regions are distributed in a large number of sites in HD; the sizes of nonhomologous regions are substantially larger than for the phages of subgroup D3112; distribution of the regions in HD is highly variable, which is characteristic of DNAs with partial homology. There is no difference between genomes of types A and B in G + C content (approx. 61-63%). Viable recombinants can be formed in crosses between phages of different genome types not only in regions with earlier revealed large DNA/DNA homology (right ends of genomes), but also in central portions of the genomes. Nevertheless, functional incompatibility of some regions of phage genomes of types A and B was demonstrated.     

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis

Thirty-three virulent and five temperate phages of Streptococcus lactis and Streptococcus cremoris were differentiated into three groups by DNA homology. A complete lack of DNA homology was demonstrated between the phage groups. Within each group, large parts of the phage genomes were homologous except for a few phages. One group consisted of five temperate and two virulent phages suggesting that virulent phages isolated during abnormal fermentations and temperate phages isolated after induction from lactic streptococcal starter cultures may be related to one another. We observed a good correlation between the grouping of phages by DNA homology and by their protein composition since within the same DNA homology group, the protein composition of a phage exhibited some similarities with that of the other phages of the group. Therefore, the DNA homologies seemed to be located, at least, in the region coding for the structural proteins. By immunoblotting, we confirmed the relatedness between the proteins of the phages belonging to the same DNA homology group. The important host range factor in bacterium-phage interactions appeared to be an unreliable criterion in determining phage taxonomy.     

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.     

Characterization of phage receptors in Streptococcus thermophilus using purified cell walls obtained by a simple protocol

A simple protocol was designed and applied to obtain Streptococcus thermophilus purified cell walls. To identify the structures involved in phage adsorption, the cell walls of two Strep. thermophilus strains were treated with sodium dodecyl sulphate and proteinase K. These treatments did not reduce the adsorption of phages CYM and 0BJ to the cell walls of Strep. thermophilus YSD10 and Strep. thermophilus BJ15, respectively. However, phage binding was reduced when the cell envelopes were treated with mutanolysin or trichloroacetic acid 5%, suggesting that the phage receptor component is part of the peptidoglycan or a polymer closely linked to it. The ability of several saccharides to inactivate both phages was also assayed. These phage inhibition experiments suggested that the phage CYM adsorbed to a component involving glucosamine and rhamnose, while glucosamine and ribose interfered with the adsorption of phage 0BJ.     

Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 10(5) PFU ml(-1)).     

Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.     

Isolation and Characterization of a Streptococcus thermophilus Plasmid Closely Related to the pMV158 Family

Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids. Fifteen S. thermophilus plasmids were divided into four groups using DNA homology. Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S. thermophilus plasmids. Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D). Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode. The only plasmid of group C, named pSMQ172, was further characterized. This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance. Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis. These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci.     

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 66(2.2)

A new bacteriophage phiK of microorganisms belonging to the genus Bordetella was isolated from cells of the earlier characterized strains 66(2-2) (1 and 2) obtained upon phage conversion of B. parapertussis 17903 cells by B. pertussis bacteriophage phi134. Bacteriophage phiK is identical to previously described Bordetella bacteriophages phiT, phi134, and phi214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage phiK is not integrated in the chromosome of B. parapertussis 17903, similar to DNA of bacteriophages phiT, phi134, and phi214 that are not integrated into B. pertussis and B. bronchiseptica chromosomes, but may be present in a small part of the bacterial population as linear plasmids. Sequences homologous to DNA of bacteriophage phiK were detected in the chromosome of strain 66(2-2) (1 and 2) and in chromosomes of all tested strains B. pertussis and B. bronchiseptica. Prophage integration in chromosomes of microorganisms of the genus Bordetella may vary in different bacterial strains and species. An assumption about abortive lysogeny of B. parapertussis bacteria for phiK phage and of B. bronchiseptica for closely related phages phiT, phi134, and phi214 has been advanced. The possibility of involvement of B. pertussis insertion sequences in the formation of the chromosomal structure in 66(2-2) convertants and in phage genomes is considered.     

DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis

Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared. DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase. DNA from all thyP-containing phages transformed thymine auxotrophs of B. subtilis SP beta lysogens to prototrophy. This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome. Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome. Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses. However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.