Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.     

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.     

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.     

Bacteriophage phi297, a new species of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> temperate phages with a mosaic genome: Potential use in phage therapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Romulus and Remus,Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus,Display Suitable Properties for Phage Therapy Applications

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.     

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.     

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

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.     

Bacteriophage-resistance mechanisms examined by transfection of Lactococcus lactis subsp. lactis 4513-5 mediated by high voltage electroporation

Summary Phage adsorption tests and transfection by electroporation were carried out to decide whether phage-resistance in Lactococcus lactis subsp. lactis strain 4513-5 is based on intracellular or extracellular mechanisms. Using high voltage (12.5 kV/cm) electroporation, untreated phage DNA was introduced into phage-sensitive and phage-resistant cells. Since phages showed low adsorption frequencies on resistant bacteria, resistance is localized in the cell wall preventing phage DNA from entering the cell. This is the only mechanism responsible for the resistance of L. lactis subsp. lactis 4513-5 against its homologous phage P4513-K12 and non-homologous phages P05M-13 and P05M-47, but not against phage P530-7 and phage P530-12. In the case of the latter two phage strains, intracellular resistance mechanisms are involved and discussed.     

Genome Sequence and Characteristics of Lrm1, a Prophage from Industrial Lactobacillus rhamnosus Strain M1

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Several new phages were obtained from the abnormally fermented broths and from the air in l-glutamic acid fermentation factory using Br. lactofermentum. These twenty-one phages were classified into five serological groups on the basis of cross-neutralization tests with homologous and heterologous antisera. Group I contained ten phages, i.e., P61, P114, P401, P465, P468I, P508, P650, P204, Ap615 and L2. Group II contained five phages, i.e., P468II, Ap85II, Ap62, Ap72 and SI. Group III contained P468III, Ap85III, Ap93 and Ap518, and groups IV and V one phage each, P4 and L1, respectively.

In view of serological similarities and of differences in the host specificity, the phages of group I are considered as host range mutants derived from a plaque type mutant, P114, of original phage P61.     

Structural Similarity and Genetic Homology between Lactobacillus casei Bacteriophages Isolated in Japan and in Finland

Three Lactobacillus casei bacteriophages, LC-Nu, PL-1, and ?FSW, were compared. Phage LC-Nu, which has not been previously characterized, originated from a local cheese plant in Finland. Phages PL-1 and ?FSW (isolated in Japan) represent the most thoroughly studied L.casei phages so far. All three phages had similar morphotypes, but still had different patterns of structural proteins, as analyzed by SDS-PAGE. The phages differed also in types of genome organization: LC-Nu and PL-1 had cohesive ends in their DNAs, and the DNA of ?FSW was circularly permuted. The initiation site and orientation of packaging of ?FSW DNA were identified. The homologies between the phage genomes were analyzed by Southern hybridization. About one-third of each phage gem me was highly homologous with other phages (homology over 85%), and two-thirds were slightly homologous (homology between 65% and 76%). DNAs from five industrial L. casei strains were also tested for homology with phage LC-Nu DNA. Phage LC-Nu related sequences were present in all the L. casei strains tested.     

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

DNA-DNA hybridizations among lactic streptococcal temperate and virulent phages belonging to distinct lytic groups

Summary Ten lactic streptococcal temperate phages and eight lactic streptococcal virulent phages classified on the basis of host range were differentiated by DNA-DNA hybridization. Virulent phages were classified in two distinct homology groups and temperate phages in a single one. In both temperate and virulent phages, no correlation was found between DNA homology groups and lytic groups. For most of the virulent phages, no DNA-DNA hybridization occurred with the temperature phages; however, partial sequence homology was found with DNAs from two virulent phages and four temperate phages.     

Alteration of Host Specificity to Lytic Bacteriophages in Streptococcus cremoris

A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.     

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.     

Study of the diversity in a group of phages of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1. These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.