Phages of non-dairy lactococci: isolation and characterization of ?L47, a phage infecting the grass isolate Lactococcus lactis ssp. cremoris DPC6860.
ABSTRACT: Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavor profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ?L47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualization of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ?L47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ?L47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs) were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage ?949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organization of ?L47 and ?949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ?L47 from ?949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5%) and a high number of predicted tRNA genes (8). Comparative genome analysis supports the conclusion that ?L47 is a new member of the 949 lactococcal phage group which currently includes the dairy ?949.
Project description:The virulent Lactococcus lactis phage 949 was isolated in 1975 from cheese whey in New Zealand. This phage is a member of the Siphoviridae family and of a rare lactococcal phage group that bears its name (949 group). It has an icosahedral capsid (79-nm diameter) and a very long noncontractile tail (length, 500 nm; width, 12 nm). It infected 7 of 59 tested L. lactis strains, a somewhat expanded host range for a rare lactococcal phage. The abortive phage infection defense mechanisms AbiQ and AbiT strongly inhibited the multiplication of phage 949, but AbiK and AbiV did not. Its double-stranded DNA (dsDNA) genome of 114,768 bp is, to date, the largest among lactococcal phages. Its GC content was calculated at 32.7%, which is the lowest reported for a lactococcal phage. Its 154 open reading frames (ORFs) share limited identity with database sequences. In addition, terminal redundancy was observed as well as the presence of six tRNAs, one group I intron, and putative recombinases. SDS-PAGE coupled with mass spectrometry identified 13 structural proteins. The genomes of the members of the 10 currently known L. lactis phage groups were used to construct a proteomic tree. Each L. lactis phage group separated into distinct genetic clusters, validating the current classification scheme. Of note, members of the polythetic P335 groups were clearly separated into subgroups.
Project description:Lactococcal bacteriophages represent one of the leading causes of dairy fermentation failure and product inconsistencies. A new member of the lactococcal 949 phage group, named WRP3, was isolated from cheese whey from a Sicilian factory in 2011. The genome sequence of this phage was determined, and it constitutes the largest lactococcal phage genome currently known, at 130,008 bp. Detailed bioinformatic analysis of the genomic region encoding the presumed initiator complex and baseplate of WRP3 has aided in the functional assignment of several open reading frames (ORFs), particularly that for the receptor binding protein required for host recognition. Furthermore, we demonstrate that the 949 phages target cell wall phospho-polysaccharides as their receptors, accounting for the specificity of the interactions of these phages with their lactococcal hosts. Such information may ultimately aid in the identification of strains/strain blends that do not present the necessary saccharidic target for infection by these problematic phages.
Project description:Dairy fermentations constitute a perfect "breeding ground" for bacteriophages infecting starter cultures, particularly strains of Lactococcus lactis. In modern fermentations, these phages typically belong to one of three groups, i.e., the 936, P335, and c2 phage groups. Traditional production methods present fewer chemical and physical barriers to phage proliferation compared to modern production systems, while the starter cultures used are typically complex, variable, and undefined. In the current study, a variety of cheese whey, animal-derived rennet, and vat swab samples from artisanal cheeses produced in Sicily were analysed for the presence of lactococcal phages to assess phage diversity in such environments. The complete genomes of 18 representative phage isolates were sequenced, allowing the identification of 10 lactococcal 949 group phages, six P087 group phages, and two members of the 936 group phages. The genetic diversity of these isolates was examined using phylogenetic analysis as well as a focused analysis of the receptor binding proteins, which dictate specific interactions with the host-encoded receptor. Thermal treatments at 63 °C and 83 °C indicate that the 949 phages are particularly sensitive to thermal treatments, followed by the P087 and 936 isolates, which were shown to be much less sensitive to such treatments. This difference may explain the relatively low frequency of isolation of the so-called "rare" 949 and P087 group phages in modern fermentations.
Project description:Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.
Project description:This report describes the morphological characterization and genome analysis of an induced prophage (PLg-TB25) from a dairy strain of Lactococcus garvieae. The phage belongs to the Siphoviridae family and its morphology is typical of other lactococcal phages. A general analysis of its genome did not reveal similarities with other lactococcal phage genomes, confirming its novelty. However, similarities were found between genes of its morphogenesis cluster and genes of Gram-positive bacteria, suggesting that this phage genome resulted from recombination events that took place in a heterogeneous microbial environment. An in silico search for other prophages in 16?L. garvieae genomes available in public databases, uncovered eight seemingly complete prophages in strains isolated from dairy and fish niches. Genome analyses of these prophages revealed three novel L. garvieae phages. The remaining prophages had homology to phages of Lactococcus lactis (P335 group) suggesting a close relationship between these lactococcal species. The similarity in GC content of L. garvieae prophages to the genomes of L. lactis phages further supports the hypothesis that these phages likely originated from the same ancestor.
Project description:Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group.IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.
Project description:Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny.
Project description: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.
Project description:P335 group phages represent the most divergent phage group infecting dairy Lactococcus lactis strains and have significant implications for the dairy processing industry. Here, we report the complete genome sequences of eight lactococcal prophages chemically induced from industrial lactococcal strains that propagate lytically on one of two laboratory strains.
Project description:Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.