Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Characterization and genomic analysis of phage asccphi28, a phage of the family Podoviridae infecting Lactococcus lactis

Bacteriophage asccphi28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccphi28 and its full genome sequence. Phage asccphi28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccphi28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccphi28 was found to be 121 +/- 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccphi28 more closely resembles streptococcal phage Cp-1 and the phi29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.     

Comparative genomic analysis of Lactococcus garvieae phage WP-2, a new member of Picovirinae subfamily of Podoviridae

To date, a few numbers of bacteriophages that infect Lactococcus garvieae have been identified, but their complete genome sequences have not yet been investigated. For the first time, herein, the complete DNA sequence of a new phage of L. garvieae (phage WP-2) is reported and analyzed. The morphological characteristics indicated that the phage had a small isometric head along with a short and non-contractile tail, suggesting that WP-2 belongs to the family Podoviridae. Bioinformatic analysis revealed that phage WP-2 can be classified as a new member of Ahjdlikevirus in the Picovirinae subfamily because it had a small dsDNA of 18,899 bp with 24 open reading frames and a protein-primed DNA polymerase. The phage nucleotide sequence and predicted protein products have been identified to share very limited evidence of homology with complete genome and proteome of other phages. To our knowledge, this is the first Ahjdlikevirus bacteriophage which can infect a member of the Lactococcus genus.     

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.     

Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis,as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.     

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.     

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.     

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.     

Resistance against Industrial Bacteriophages Conferred on Lactococci by Plasmid pAJ1106 and Related Plasmids

Plasmid pAJ1106 and its deletion derivative, plasmid pAJ2074, conferred lactose-fermenting ability (Lac) and bacteriophage resistance (Hsp) at 30°C to Lac⁻ proteinase (Prt)-negative Lactococcus lactis subsp. lactis and L. lactis subsp. lactis var. diacetylactis recipient strains. An additional plasmid, pAJ331, isolated from the original source strain of pAJ1106, retained Hsp and conjugative ability without Lac. pAJ331 was conjugally transferred to two L. lactis subsp. lactis and one L. lactis subsp. cremoris starter strains. The transconjugants from such crosses acquired resistance to the phages which propagated on the parent recipient strains. Of 10 transconjugant strains carrying pAJ1106 or one of the related plasmids, 8 remained insensitive to phages through five activity test cycles in which cultures were exposed to a large number of industrial phages at incubation temperatures used in lactic casein manufacture. Three of ten strains remained phage insensitive through five cycles of a cheesemaking activity test in which cultures were exposed to approximately 80 different phages through cheesemaking temperatures. Three phages which propagated on transconjugant strains during cheesemaking activity tests were studied in detail. Two were similar (prolate) in morphology and by DNA homology to phages which were shown to be sensitive to the plasmid-encoded phage resistance mechanism. The third phage was a long-tailed, small isometric phage of a type rarely found in New Zealand cheese wheys. The phage resistance mechanism was partially inactivated in most strains at 37°C.     

Identification of Lactococcus lactis Genes Required for Bacteriophage Adsorption

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that 645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and 645 that showed no homology in the C-terminal part.     

Sequence Analysis and Molecular Characterization of the Lactococcus lactis Temperate Bacteriophage BK5-T

The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3′ single-stranded overhang with the sequence 5′-CACACACATAGG-3′. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).     

Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a G+C content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ΦHAP-1.     

Genome Sequence of the Bacteriocin-Producing Strain Lactococcus garvieae DCC43

This work describes the draft genome sequence of Lactococcus garvieae DCC43. The 2.2-Mb draft genome contains 2,227 predicted protein-coding genes, among which is a region encoding the bacteriocin garvicin ML. No antibiotic resistance genes or capsule-related virulence genes were identified. Two plasmid replication regions indicate that this strain likely contains plasmids. Comparative genomics suggests that this strain displays a high degree of sequence variation from the previously sequenced L. garvieae strains.     

Lactococcus garvieae: Where Is It From? A First Approach to Explore the Evolutionary History of This Emerging Pathogen

The population structure and diversity of Lactococcus garvieae, an emerging pathogen of increasing clinical significance, was determined at both gene and genome level. Selected lactococcal isolates of various origins were analyzed by a multi locus sequence typing (MLST). This gene-based analysis was compared to genomic characteristics, estimated through the complete genome sequences available in database. The MLST identified two branches containing the majority of the strains and two branches bearing one strain each. One strain was particularly differentiated from the other L. garvieae strains, showing a significant genetic distance. The genomic characteristics, correlated to the MLST-based phylogeny, indicated that this “separated strain” appeared first and could be considered the evolutionary intermediate between Lactococcus lactis and L. garvieae main clusters. A preliminary genome analysis of L. garvieae indicated a pan-genome constituted of about 4100 genes, which included 1341 core genes and 2760 genes belonging to the dispensable genome. A total of 1491 Clusters of Orthologous Genes (COGs) were found to be specific to the 11 L. garvieae genomes, with the genome of the “separated strain” showing the highest presence of unique genes.     

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.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems

Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage.