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.     

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

Background

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.

Results

Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.

Conclusions

SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-831) contains supplementary material, which is available to authorized users.     

Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.     

Acid stress response in Helicobacter pylori

Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects phiP335 itself and phiQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined.     

Monoclonal Antibodies Raised against Native Major Capsid Proteins of Lactococcal c2-Like Bacteriophages

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.     

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.     

Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage sensitivity of a particular lactococcal strain and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages while also partially explaining the molecular reasons behind the observed narrow host range of such phages.     

Structure,Adsorption to Host,and Infection Mechanism of Virulent Lactococcal Phage p2

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k_off values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.     

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.     

Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains

A set of 83 lytic dairy bacteriophages (phages) infecting flavor-producing mesophilic starter strains of the Leuconostoc genus was characterized, and the first in-depth taxonomic scheme was established for this phage group. Phages were obtained from different sources, i.e., from dairy samples originating from 11 German dairies (50 Leuconostoc pseudomesenteroides [Ln. pseudomesenteroides] phages, 4 Ln. mesenteroides phages) and from 3 external phage collections (17 Ln. pseudomesenteroides phages, 12 Ln. mesenteroides phages). All phages belonged to the Siphoviridae family of phages with isometric heads (diameter, 55 nm) and noncontractile tails (length, 140 nm). With the exception of one phage (i.e., phage ΦLN25), all Ln. mesenteroides phages lysed the same host strains and revealed characteristic globular baseplate appendages. Phage ΦLN25, with different Y-shaped appendages, had a unique host range. Apart from two phages (i.e., phages P792 and P793), all Ln. pseudomesenteroides phages shared the same host range and had plain baseplates without distinguishable appendages. They were further characterized by the presence or absence of a collar below the phage head or by unique tails with straight striations. Phages P792 and P793 with characteristic fluffy baseplate appendages could propagate only on other specific hosts. All Ln. mesenteroides and all Ln. pseudomesenteroides phages were members of two (host species-specific) distinct genotypes but shared a limited conserved DNA region specifying their structural genes. A PCR detection system was established and was shown to be reliable for the detection of all Leuconostoc phage types.     

Evolution of Lactococcus lactis Phages within a Cheese Factory

We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3′ overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.The manufacture of cheeses requires the inoculation of carefully selected bacterial cultures, known as starter cultures, at concentrations of at least 10⁷ live bacteria per ml of heat-treated milk. The purpose of this process is to control the fermentation and to obtain high-quality fermented products (29). Starter cultures are a combination of lactic acid bacteria (LAB), of which one of the most important species is Lactococcus lactis. L. lactis is a low-GC gram-positive bacterium used to metabolize lactose into lactic acid during the production of several cheese varieties. Because large amounts of lactococcal cells are cultivated each day in large-scale fermentation vats and because these cells are susceptible to bacteriophage infection, it is not surprising that most cheese factories have experienced problems with phage contamination (13). Even a single phage infecting a starter strain is enough to begin a chain reaction that can eventually inhibit bacterial growth and cause production delays, taste and texture variations, and even complete fermentation failures (1, 29).Phage infections are unpredictable in food fermentations. Their presence and persistence in a dairy factory can be explained in many ways. First, raw milk can introduce new phages into an industrial plant (25). Madera et al. (22) also reported that newly isolated lactococcal phages were more resistant to pasteurization. Whey, a liquid by-product of cheese manufacturing, is another reservoir that can spread phages in a factory environment (25). Airborne phage dissemination may also be important since concentrations of up to 10⁶ PFU/m³ have been observed close to a functional whey separation tank (32).For decades, the dairy industry has been working to curtail the propagation of virulent phages using a variety of practical strategies, including, among others, sanitation, optimized factory design, air filtration units, rotation of bacterial strains, and the use of phage resistance systems (13). Yet new virulent phages emerge on a regular basis. Indeed, large-scale industrial milk fermentation processes can be slowed down by virulent phages of the Caudovirales order. Members of three lactococcal phage groups, namely, 936, c2, and P335, are mostly found in dairy plants. The 936-like phages are by far the most predominant worldwide (3, 18, 22, 27).Phages of the 936 group have a double-stranded DNA genome and possess a long noncontractile tail connected to a capsid with icosahedral symmetry characteristic of the Siphoviridae family. Currently, six complete phage genomes of the lactococcal 936 group are available in public databases, including sk1 (6), bIL170 (10), jj50, 712, P008 (23), and bIBB29 (16). Their comparative analysis revealed a conserved gene organization despite being isolated from different countries. Most of the differences have been observed in the early gene module, where insertions, deletions, and point mutations likely occurred (16, 23). Moreover, it is assumed that these phages can also exchange DNA through recombination with other bacterial viruses present in the same ecosystem.Because new members of this lactococcal phage group are regularly isolated, a better understanding of their evolution is warranted to better control them. A cheese factory is a particular man-made niche where rapidly growing bacterial strains encounter ubiquitous phages. Such active environments provide ample opportunities for phage evolution, especially to dodge phage resistance mechanisms that may be present in host cells. Nonetheless, the evolutionary dynamics that shape the diversity of lactococcal phage populations are still not well understood.In this study, we analyzed the genome and structural proteome of six 936-group phages (SL4, CB13, CB14, CB19, CB20, and GR7) that infected the same L. lactis strains and were isolated over a 9-year period from a cheese factory.     

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.     

Biodiversity and classification of lactococcal phages

For this study, an in-depth review of the classification of Lactococcus lactis phages was performed. Reference phages as well as unclassified phages from international collections were analyzed by stringent DNA-DNA hybridization studies, electron microscopy observations, and sequence analyses. A new classification scheme for lactococcal phages is proposed that reduces the current 12 groups to 8. However, two new phages (Q54 and 1706), which are unrelated to known lactococcal phages, may belong to new emerging groups. The multiplex PCR method currently used for the rapid identification of phages from the three main lactococcal groups (936, c2, and P335) was improved and tested against the other groups, none of which gave a PCR product, confirming the specificity of this detection tool. However, this method does not detect all members of the highly diverse P335 group. The lactococcal phages characterized here were deposited in the Félix d'Hérelle Reference Center for Bacterial Viruses and represent a highly diverse viral community from the dairy environment.     

Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72°C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Modular structure of the receptor binding proteins of Lactococcus lactis phages. The RBP structure of the temperate phage TP901-1

Lactococcus lactis is a gram-positive bacterium widely used by the dairy industry. Several industrial L. lactis strains are sensitive to various distinct bacteriophages. Most of them belong to the Siphoviridae family and comprise several species, among which the 936 and P335 are prominent. Members of these two phage species recognize their hosts through the interaction of their receptor-binding protein (RBP) with external cell wall saccharidices of the host, the "receptors." We report here the 1.65 A resolution crystal structure of the RBP from phage TP901-1, a member of the P335 species. This RBP of 163 amino acids is a homotrimer comprising three domains: a helical N terminus, an interlaced beta-prism, and a beta-barrel, the head domain (residues 64-163), which binds a glycerol molecule. Fluorescence quenching experiments indicated that the RBP exhibits high affinity for glycerol, muramyl-dipeptide, and other saccharides in solution. The structural comparison of this RBP with that of lactococcal phage p2 RBP, a member of the 936 species (Spinelli, S., Desmyter, A., Verrips, C. T., de Haard, J. W., Moineau, S., and Cambillau, C. (2006) Nat. Struct. Mol. Biol. 13, 85-89) suggests a large extent of modularity in RBPs of lactococcal phages.     

Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages

Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.The first sequenced genome of a phage infecting Lactococcus lactis (bIL67) was reported in 1994 (57). Its genomic characterization was performed with the prospect of a better understanding of lactococcal phage biology. L. lactis is a Gram-positive bacterium added to milk to produce an array of fermented dairy products. In this human-made environment, substantial amounts of lactococcal cells are cultivated on a daily basis in large fermentation vats, and these added cells randomly encounter virulent phages present in heat-treated but nonsterile milk. Moreover, it is widely acknowledged that the increased use of the same bacterial strains within existing dairy facilities inevitably leads to milk fermentation failures due to the multiplication of virulent phages. This biotechnological problem reduces yields and lowers the quality of fermented products (51).Over 700 lactococcal phage isolates have been reported in the literature (3). To date, more than 25 complete genome sequences of lactococcal phages are publicly available in the NCBI database, and the sequencing of others is under way. These numbers indicate that Lactococcus phages are among the most studied of the bacterial viruses. All lactococcal phages belong to the order Caudovirales and are included within two families according to their tail morphology: the Siphoviridae (long noncontractile tail [most lactococcal phages]) and the Podoviridae (short noncontractile tail [few lactococcal phages]) (14). Currently, phages infecting L. lactis strains have been divided into 10 genetically distinct groups (14). The complete genomic sequence is available for at least one representative of 8 of the groups.Early sequencing efforts concentrated on the genomes of lactococcal phages belonging to the 936, c2, and P335 groups (Siphoviridae), because members of these groups were regularly isolated in dairy plants (8, 36, 50). PCR-based methods were also devised to rapidly classify these phages (41). These Siphoviridae phages pose a significant risk to the dairy industry, and their characterization is important for developing adapted antiphage strategies to limit their propagation and evolution.In recent years, representatives of the less recognized lactococcal phage groups have been characterized, including phages Q54 (22), KSY1 (13), 1706 (23), asccφ28 of the P034 group (39), and P087 (63). Their molecular characterizations were aimed at understanding why some phage groups (936, c2, and P335) predominate while the others have remained marginal, at best. However, it was recently reported that P034-like phages may be emerging in certain regions (52). Genomic and microbiological analyses indicated that members of these rare phage groups were likely the result of recombination between different lactococcal phages and phages infecting other Gram-positive bacteria, and they may not be fit to multiply rapidly in milk. For example, lactococcal phage 1706 shares similarities with Ruminococcus and Clostridium prophages (23). Similarly, L. lactis phage P087 structural proteins share identity with gene products found in a prophage in the Enterococcus faecalis genome (63). It was also shown previously that lactococcal phage asccφ28 was related to Streptococcus pneumoniae phage Cp-1 and Bacillus subtilis φ29-like phages (39). It was suggested that phages 1706, asccφ28, and P087 acquired a receptor-binding protein complex from another lactococcal phage that enabled them to infect a L. lactis host.Here, we report the complete genome sequence and analysis of phage 1358, a virulent representative of the 9th lactococcal phage group.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.