Molecular taxonomy of Lactobacillus phages

Forty-eight strains of lactobacilli used as starter strains in the dairy industry were examined for lysogeny after treatment with mitomycin C. Two strains of L. delbrueckii subsp. bulgaricus were able to produce active phages. These temperate phages as well as 4 virulent phages isolated during abnormal fermentations were compared to a previously characterized phage mv4 which is temperate. All these phages were shown to be partially homologous by DNA-DNA hybridization. Genes that code for viral proteins seem to be well conserved since 2 major virion polypeptides of 18 (or 19) kD and 34 kD could be detected in the protein composition of each phage. Immunoblotting studies of the 7 phages using serum raised against phage mv4 confirmed that the proteins of the different phages were related. All these phages can be classified in the previously constituted group a, which now comprises 4 temperate and 15 virulent phages. These results show that some virulent phages appearing during abnormal fermentations and some temperate phages isolated by appearing during abnormal fermentations and some temperate phages isolated by induction of starter strains can be closely related genetically. Five virulent phages of L. helveticus were also compared according to their restriction pattern and their DNA homology. They were shown to be related to one another, but unrelated to phages of other lactic acid bacteria species.     

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.     

Multiplex PCR for detection and identification of lactococcal bacteriophages

Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.     

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.     

A new phage typing scheme forProteus mirabilis andProteus vulgaris strains

A new bacteriophage typing set, composed of 22 phages, was established as a tool for differentiation ofProteus strains. All the phages were tailed and included 4 morphological types (A1, A2, B1 and C1). They were classified into the familiesMyoviridae, Siphoviridae andPodoviridae. From the set, 19 phages had double-stranded DNA and 3 were single-stranded DNA phages.     

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.     

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.     

Comparison of genomes of new gigantic Pseudomonas aeruginosa phages from native populations from different regions

To study the genome diversity of bacteriophages from geographically distant natural populations, new giant phi KZ-like Pseudomonas aeruginosa phages isolated in two different regions were compared with earlier known phages of three species (phi KZ, Lin68, EL). A broad spectrum of lytic activity was demonstrated for all phi KZ-like phages. Phages of the phi KZ species proved to be common in natural populations of various regions, while IL- and Lin68-related phages were extremely rare. Most phi KZ-related phages had unique DNA restriction patterns, but the differences between these were only minor, and the genomes did not contain nonhomologous fragments. The spectrum of capsid polypeptides proved to be conserved in each species, and was proposed as a character necessary and sufficient for express classification of phages with an accuracy of species. Phages isolated in different geographical regions showed no substantial difference. Some phages only slightly differing in DNA restriction pattern from phi KZ may be used to study the origin of phi KZ genes coding for orthologs of proteins of unrelated species (other phages, pathogenic bacteria, eukaryotes).     

Restriction/Modification systems and restriction endonucleases are more effective on lactococcal bacteriophages that have emerged recently in the dairy industry

Recently, eight lytic small isometric-headed bacteriophages were isolated from cheese-manufacturing plants throughout North America. The eight phages were different, but all propagated on one strain, Lactococcus lactis NCK203. On the basis of DNA homology, they were classified in the P335 species. Digestion of their genomes in vitro with restriction enzymes resulted in an unusually high number of type II endonuclease sites compared with the more common lytic phages of the 936 (small isometric-headed) and c2 (prolate-headed) species. In vivo, the P335 phages were more sensitive to four distinct lactococcal restriction and modification (R/M) systems than phages belonging to the 936 and c2 species. A significant correlation was found between the number of restriction sites for endonucleases (purified from other bacterial genera) and the relative susceptibility of phages to lactococcal R/M systems. Comparisons among these three phage species indicate that the P335 species may have emerged most recently in the dairy industry.     

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).     

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.     

Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.     

Effect of exopolysaccharides on phage-host interactions in Lactococcus lactis

In this study, we report that Lactococcus lactis strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps(+) strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps(-) L. lactis strains. The host range of these phages was determined with 19 Lactococcus strains, including 7 Eps(+) and 12 Eps(-) cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps(+) strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps(+) strain SMQ-420. The five other Eps(+) strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps(-) strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps(+) strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the eps operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype.     

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.     

Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata.

Thirty-three wild type strains of Rhodopseudomonas capsulata were examined for ability to engage in genetic recombination through mediation by "gene transfer agent" (GTA) particles. The genetic exchange assays were based on capacity of strains to produce or receive GTA required for restoration of photosynthetic growth competence to a non-photosynthetic "white" mutant or for acquisition of resistance to rifampicin. A majority of the strains could either produce or receive GTA, and it was demonstrated that the agent is species specific. Possible relations between GTA and bacteriophages or bacteriocins were investigated. Sixteen types of virulent phages active on Rps. capsulata were isolated and their host ranges determined. Tests for transduction by the phages gave uniformly negative results. The viruses showed strict species specificity, but there was no apparent correlation between capacity of the Rps. capsulata strains to donate or receive GTA and susceptibility to the phages. A comparable survey disclosed that most of the bacterial strains were sensitive to or capable of producing bacteriocins; the latter also appear to be unrelated to GTA activity. The collection of bacterial strains was also screened for detection of lysogenic properties. None of the isolates is a "true" lysogen, but phages were detected in cultures of two strains, which may be "phage carriers" or pseudolysogens.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.     

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage φIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, φIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.     

Search for destruction factors of bacterial biofilms: Comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products

Comparison of Pseudomonas putida group of phages attributed to five species (af, ?15, ?27, ?2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.     

Gene transfer agents,bacteriophages, and bacteriocins of Rhodopseudomonas capsulata

Thirty-three wild type strains of Rhodopseudomonas capsulata were examined for ability to engage in genetic recombination through mediation by “gene transfer agent” (GTA) particles. The genetic exchange assays were based on capacity of strains to produce or receive GTA required for restoration of photosynthetic growth competence to a non-photosynthetic “white” mutant or for acquisition of resistance to rifampicin. A majority of the strains could either produce or receive GTA, and it was demonstrated that the agent is species specific. Possible relations between GTA and bacteriophages or bacteriocins were investigated. Sixteen types of virulent phages active on Rps. capsulata were isolated and their host ranges determined. Tests for transduction by the phages gave uniformly negative results. The viruses showed strict species specificity, but there was no apparent correlation between capacity of the Rps. capsulata strains to donate or receive GTA and susceptibility to the phages. A comparable survey disclosed that most of the bacterial strains were sensitive to or capable of producing bacteriocins; the latter also appear to be unrelated to GTA activity. The collection of bacterial strains was also screened for detection of lysogenic properties. None of the isolates is a “true” lysogen, but phages were detected in cultures of two strains, which may be “phage carriers” or pseudolysogens.