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The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities., Competing Interests: P.P.D.W., I.M.H.V.R., and N.N.M.E.V.P. work for dsm-firmenich.

Lactococcus phages that belong to the genus Ceduovirus are among the three most frequently isolated phage groups infecting Lactococcus lactis starter strains in dairy plants. In this study, we characterized virulent Lactococcus phage BIM BV-114 isolated from industrial cheese brine in Belarus and identified as Ceduovirus. The bacteriophage demonstrated a relatively short lytic cycle (latent period of 23 ± 5 min, lysis time of 90 ± 5 min), high thermal stability (inactivation after 7 min at 95 °C in skimmed milk) and tolerance to UV radiation (inactivation time - 15 min), indicating adaptation for better persistence in dairy facilities. The genome of the phage BIM BV-114 (21 499 bp; 37 putative open reading frames) has a similar organization to that of other Ceduovirus phages. RLf1_00140 and RLf_00050 gene products, found in the early genes region, may be involved in the sensitivity of phage to the lactococcal abortive infection mechanisms AbiV and AbiQ, respectively. Furthermore, nucleotide deletion, observed in the middle region of the gene encoding putative tape measure protein (RLf1_00300), is possibly responsible for increased thermal tolerance of phage BIM BV-114. Together, these findings will contribute to a better knowledge of virulent Lactococcus phages and the development of effective methods of their control for dairy technologies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an in vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced in vitro, and analysed in SDS-PAGE, giving a 42-kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by in vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Bacteriophages infecting dairy starter bacteria are a leading cause of milk fermentation failure and strategies to reduce the risk of phage infection in dairy settings are demanded. Along with dairy starters, bacteriocin producers (protective cultures) or the direct addition of bacteriocins as biopreservatives may be applied in food to extend shelf-life. In this work, we have studied the progress of infection of Lactococcus cremoris MG1363 by the phage sk1, in the presence of three bacteriocins with different modes of action: nisin, lactococcin A (LcnA), and lactococcin 972 (Lcn972). We aimed to reveal putative bacteriocin-phage interactions (BaPI) that could be detrimental and increase the risk of fermentation failure due to phages. Based on infections in broth and solid media, a synergistic effect was observed with Lcn972. This positive sk1-Lcn972 interaction could be correlated with an increased burst size. sk1-Lcn972 BaPI occurred independently of a functional SOS and cell envelope stress response but was lost in the absence of the major autolysin AcmA. Furthermore, BaPI was not exclusive to the sk1-Lcn972 pairing and could be observed with other phages and lactococcal strains. Therefore, bacteriocins may facilitate phage predation of dairy lactococci and their use should be carefully evaluated., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Background: Staphylococcus aureus is an opportunistic Gram-positive bacterium that can form biofilm and become resistant to many types of antibiotics. The treatment of multi-drug resistant Staphylococcus aureus (MDRSA) infection is difficult since it possesses multiple antibiotic-resistant mechanisms. Endolysin and virion-associated peptidoglycan hydrolases (VAPGH) enzymes from bacteriophage have been identified as potential alternative antimicrobial agents. This study aimed to assess the ability of Lactococcus lactis NZ9000 secreting endolysin and VAPGH from S. aureus bacteriophage 88 to inhibit the growth of S. aureus PS 88, a MDRSA., Method: Endolysin and VAPGH genes were cloned and expressed in L. lactis NZ9000 after fusion with the SPK1 signal peptide for secretion. The recombinant proteins were expressed and purified, then analyzed for antimicrobial activity using plate assay and turbidity reduction assay. In addition, the spent media of the recombinant lactococcal culture was analyzed for its ability to inhibit the growth of the S. aureus PS 88., Results: Extracellular recombinant endolysin (Endo88) and VAPGH (VAH88) was successfully expressed and secreted from L. lactis which was able to inhibit S. aureus PS 88, as shown by halozone formation on plate assays as well as inhibition of growth in the turbidity reduction assay. Moreover, it was observed that the spent media from L. lactis NZ9000 expressing Endo88 and VAH88 reduced the viability of PS 88 by up to 3.5-log reduction with Endo88 being more efficacious than VAH88. In addition, Endo88 was able to lyse all MRSA strains tested and Staphylococcus epidermidis but not the other bacteria while VAH88 could only lyse S. aureus PS 88., Conclusion: Recombinant L. lactis NZ9000 expressing phage 88 endolysin may be potentially developed into a new antimicrobial agent for the treatment of MDRSA infection., Competing Interests: The authors declare there are no competing interests., (©2022 Chandran et al.)

Prophages are widely present in Lactococcus lactis, a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid and prophage free, MG1363 carries two complete prophages, TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the "lytic TP712 lysogens" which had remained unnoticed. CAP is able to be excised after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression in trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work that generated the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor, L. lactis NCDO712, and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the "presequencing" era. IMPORTANCE Prophages are bacterial viruses that integrate into the chromosomes of bacteria until an environmental trigger induces their lytic cycle, ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we discovered that two temperate phages, TP712 and CAP, infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the lysogens formerly thought to be single TP712 lysogens that had remained a mystery for many years.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Non-model bacteria like Pseudomonas putida, Lactococcus lactis and other species have unique and versatile metabolisms, offering unique opportunities for Synthetic Biology (SynBio). However, key genome editing and recombineering tools require optimization and large-scale multiplexing to unlock the full SynBio potential of these bacteria. In addition, the limited availability of a set of characterized, species-specific biological parts hampers the construction of reliable genetic circuitry. Mining of currently available, diverse bacteriophages could complete the SynBio toolbox, as they constitute an unexplored treasure trove for fully adapted metabolic modulators and orthogonally-functioning parts, driven by the longstanding co-evolution between phage and host.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis , controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15 N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R 1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance., Competing Interests: The authors declare no competing interest.

Virulent phages infecting L. lactis , an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca 2+ . However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

The lactococcal virulent phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects Lactococcus lactis MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.

A functional genetic switch from the lactococcal bacteriophage TP901-1, deciding which of two divergently transcribing promoters becomes most active and allows this bi-stable decision to be inherited in future generations requires a DNA region of less than 1 kb. The fragment encodes two repressors, CI and MOR, transcribed from the P R and P L promoters respectively. CI can repress the transcription of the mor gene at three operator sites (O R , O L , and O D ), leading to the immune state. Repression of the cI gene, leading to the lytic (anti-immune) state, requires interaction between CI and MOR by an unknown mechanism, but involving a CI:MOR complex. A consensus for putative MOR binding sites (O M sites), and a common topology of three O M sites adjacent to the O R motif was here identified in diverse phage switches that encode CI and MOR homologs, in a search for DNA sequences similar to the TP901-1 switch. The O R site and all putative O M sites are important for establishment of the anti-immune repression of P R , and a putative DNA binding motif in MOR is needed for establishment of the anti-immune state. Direct evidence for binding between CI and MOR is here shown by pull-down experiments, chemical crosslinking, and size exclusion chromatography. The results are consistent with two possible models for establishment of the anti-immune repression of cI expression at the P R promoter.

Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails' distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host's surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae .

To date, a number of bacteriophages that infect Lactococcus garvieae isolated from marine fish have been identified. However, the evolutionary insight between L. garvieae phages and other viral community have not yet been immersedly investigated. In this study, completed genomic sequence of phage PLgY-30 was obtained, a comparative analysis of three lytic phages, which have been using for phage typing and treatment of L. garvieae infecting marine fish, is conducted. The results revealed that the genomes of lytic phages specific for L. garvieae isolated from diseased marine fish share a high level of homology and almost all proteins are conserved. At genome level, no similarity was detected for either PLgY-30 or PLgY-16, while PLgW-1 shares only very limited homology (1%) with other sequences in Genbank database. In addition, the function of only 35% of ORFs in the PLgY-30 phage genomes could be predicted, demonstrating that it is novel phage. At protein level, lytic phage proteins shared a significant similarity to various proteins of global phage species isolated from dairy fermentation facilities that utilize L. lactis as a primary starter culture, called the 936 phage group. Genome organization and architecture of three lytic phages are also similar to that of the 936 phage group. To our knowledge, this is the first time lytic bacteriophages infecting L. garvieae from marine fish were characterized to genome level., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system., Results: The constructed integration system comprises of a lactococcal promoter (P nisA or P 170 ), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP 344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P 170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA., Conclusion: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O -acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria., Competing Interests: The authors declare no conflict of interest.

Lactococcus lactis is a Gram-positive bacterium widely used as a starter culture for the production of different dairy products, especially a large variety of cheeses. Infection of lactococcal starter cultures by bacteriophages is one of the major causes of fermentation failure and often leads to production halt. Lactococcal bacteriophages belonging to the c2, 936, and P335 species are the most commonly isolated in dairy plants and have been extensively investigated in the past three decades. Information regarding bacteriophages belonging to less commonly isolated species is, on the other hand, less extensive, although these phages can also contribute to starter culture infection. Here, we report the nucleotide sequence of the newly isolated L. lactis phage CHPC971, belonging to the rare 1706 species of lactococcal phages. We investigated the nature of the host receptor recognized by the phage and collected evidence that strongly suggests that it binds to a specific sugar moiety in the cell wall pellicle of its host. An in silico analysis of the genome of phage CHPC971 identified the hypothetical genes involved in receptor binding. IMPORTANCE Gathering information on how lactococcal bacteriophages recognize their host and proliferate in the dairy environment is of vital importance for the establishment of proper starter culture rotation plans and to avoid fermentation failure and consequent great economic losses for dairy industries. We provide strong evidence on the type of receptor recognized by a newly isolated 1706-type lactococcal bacteriophage, increasing knowledge of phage-host interactions relevant to dairying. This information can help to prevent phage infection events that, so far, are hard to predict and avoid., (Copyright © 2019 American Society for Microbiology.)

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural "bricks" to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

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.

Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions., (© 2019 Lemay et al.)

With increasing numbers of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and associated functionality of phage adhesion devices. Recently, decorations have been reported in the tail and neck passage structures of members of the so-called 936 group of lactococcal siphophages. In the current report, using bioinformatic analysis we identified a conserved carbohydrate binding module (CBM) among many of the virion baseplate Dit components, in addition to the CBM present in the 'classical' receptor binding proteins (RBPs). We observed that, within these so-called 'evolved' Dit proteins, the identified CBMs have structurally conserved folds, yet can be grouped into four distinct classes. We expressed such modules in fusion with GFP, and demonstrated their binding capability to their specific host using fluorescent binding assays with confocal microscopy. We detected evolved Dits in several phages infecting various Gram-positive bacterial species, including mycobacteria. The omnipresence of CBM domains in siphophages indicates their auxiliary role in infection, as they can assist in the specific recognition of and attachment to their host, thus ensuring a highly efficient and specific phage-host adhesion process as a prelude to DNA injection., (© 2018 John Wiley & Sons Ltd.)

Siphoviridae of the lactococcal 936 group are the most commonly encountered bacteriophages in the dairy processing environment. The 936 group phages possess a discrete baseplate at the tip of their tail-a complex harbouring the Receptor Binding Protein (RBP) which is responsible for host recognition and attachment. The baseplate-encoding region is highly conserved amongst 936 phages, with 112 of 115 publicly available phages exhibiting complete synteny. Here, we detail the three exceptions (Phi4.2, Phi4R15L, and Phi4R16L), which differ from this genomic architecture in possessing an apparent second RBP-encoding gene upstream of the "classical" rbp gene. The newly identified RBP possesses an elongated neck region relative to currently defined 936 phage RBPs and is genetically distinct from defined 936 group RBPs. Through detailed characterisation of the representative phage Phi4.2 using a wide range of complementary techniques, we demonstrated that the above-mentioned three phages possess a complex and atypical baseplate structure. Furthermore, the presence of both RBPs in the tail tip of the mature virion was confirmed, while the anticipated host-binding capabilities of both proteins were also verified.

Background: In complex microbial ecosystems such as the marine environment, the gastrointestinal tract, but also in mixed culture fermentations, bacteriophages are frequently found to be a part of the microbial community. Moreover, prophages or prophage-like elements are frequently identified in sequenced bacterial genomes. The mixed undefined starter cultures represent an ecosystem which is shaped by long term evolution under relatively defined environmental conditions and provides an interesting model to study co-evolution of phages and their hosts as well as the impact of diversity on microbial community stability., Results: In the present study we investigated the presence, identity and behaviour of prophages in lactococci being part of a complex cheese starter culture. Genome analysis of representative strains of the 7 genetic lineages of Lactococcus lactis constituting the culture indicated the presence of prophages in all strains. Exposure of potential lysogens to mitomycin C confirmed the release of ~ 10 10 ·ml - 1 phage particles from all tested strains. Furthermore, phages were also released in substantial amounts due to spontaneous induction: more than 10 8 ·ml - 1 phage particles were present in cultures under non-inducing conditions. This observation suggests continuous release of phage particles by the lactococci. The released bacteriophages exhibited an unusual morphology. For most strains tested, tailless icosahedral phage heads were found. The competitive advantage of lysogens compared to their cured derivatives and their high abundance in the culture suggests that the released tailless bacteriophages play an important role in the ecosystem., Conclusions: The results of this study indicate that chromosomal genetic elements are active participants in the stable complex microbial community of the starter culture. We show that prophages are abundant in such a community, are produced continuously in large amounts and, despite the huge metabolic burden imposed on the cells by phage particle production, provide a selective advantage to the host.

Bacteriophage infection of Lactococcus species can cause serious disruption of dairy fermentation processes. The most common isolates from the dairy environment are Siphoviridae lytic 936-type phages. To gain specific knowledge about this group of phages in Polish dairies, we examined 90 isolates from 8 different locations. Based on restriction fragment length polymorphism analysis, coupled with physiological and molecular studies, the isolated phages were divided into 8 distinct groups. Whole-genome sequencing of single representatives from each phage group provided data about their biology and genetic composition. The phages present an overall conserved genome organization. High sequence homology to another Polish isolate, Lactococcus phage bIBB29, indicates their close phylogenetic relatedness to this strain. Such similarity may be suggestive of a general genome conservation among phages persisting in Polish dairies. Comparative genome analyses with other 936-type phages revealed several discriminative traits, including the presence and position of HNH endonuclease genes, varying number of orfs in the early gene region, and a putative TpeX gene. Interestingly, host range of the sequenced phages was restricted to L. lactis subsp. lactis biovar. diacetylactis strains. The results provide new data regarding phages present in the Polish dairy environment and permit analysis of their biology, genome composition and relatedness to other Lactococcus 936-type phages.

The inherent ability of bacteriophages (phages) to infect specific bacterial hosts makes them ideal candidates to develop into antimicrobial agents for pathogen-specific remediation in food processing, biotechnology, and medicine (e.g., phage therapy). Conversely, phage contaminations of fermentation processes are a major concern to dairy and bioprocessing industries. The first stage of any successful phage infection is adsorption to a bacterial host cell, mediated by receptor-binding proteins (RBPs). As the first point of contact, the binding specificity of phage RBPs is the primary determinant of bacterial host range, and thus defines the remediative potential of a phage for a given bacterium. Co-evolution of RBPs and their bacterial receptors has forced endless adaptation cycles of phage-host interactions, which in turn has created a diverse array of phage adsorption mechanisms utilizing an assortment of RBPs. Over the last decade, these intricate mechanisms have been studied intensely using electron microscopy and X-ray crystallography, providing atomic-level details of this fundamental stage in the phage infection cycle. This review summarizes current knowledge surrounding the molecular basis of host interaction for various socioeconomically important Gram-positive targeting phage RBPs to their protein- and saccharide-based receptors. Special attention is paid to the abundant and best-characterized Siphoviridae family of tailed phages. Unravelling these complex phage-host dynamics is essential to harness the full potential of phage-based technologies, or for generating novel strategies to combat industrial phage contaminations.

Lactococcus lactis is a lactic acid bacterium that is intensively and globally exploited in commercial dairy food fermentations. Though the presence of prophages in lactococcal genomes is widely reported, only limited studies pertaining to the stability of prophages in lactococcal genomes have been performed. The current study reports on the complete genome exploration of thirty lactococcal strains for the presence of potentially intact prophages, so as to assess their genomic diversity and the associated risk or benefit of harbouring such prophages. Genomic predictions partnered with mitomycin C inductions and flow cytometric analysis of the induced cell lysates confirmed that only four strains consistently produced intact phage particles, thus indicating a relatively low risk associated with prophage induction in the fermentation setting. Our analysis revealed the widespread presence of putative phage-resistance systems encoded by lactococcal prophages, thus highlighting the potential benefits for host fitness. Many of the identified lactococcal prophages belong to the so-called P335 phage group, while a large group of phage remnants bear similarity to members of the 936 phage group. The P335 phage group was recently shown to encompass four distinct genetic lineages. Our study identified an additional lineage, thus expanding the diversity of this industrially significant phage group., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The evolution of asexual organisms is driven not only by the inheritance of genetic modification but also by the acquisition of foreign DNA. The contribution of vertical and horizontal processes to genome evolution depends on their rates per year and is quantified by the ratio of recombination to mutation. These rates have been estimated for bacteria; however, no estimates have been reported for phages. Here, we delineate the contribution of mutation and recombination to dsDNA phage genome evolution. We analyzed 34 isolates of the 936 group of Siphoviridae phages using a Lactococcus lactis strain from a single dairy over 29 years. We estimate a constant substitution rate of 1.9 × 10-4 substitutions per site per year due to mutation that is within the range of estimates for eukaryotic RNA and DNA viruses. The reconstruction of recombination events reveals a constant rate of five recombination events per year and 4.5 × 10-3 nucleotide alterations due to recombination per site per year. Thus, the recombination rate exceeds the substitution rate, resulting in a relative effect of recombination to mutation (r/m) of ∼24 that is homogenous over time. Especially in the early transcriptional region, we detect frequent gene loss and regain due to recombination with phages of the 936 group, demonstrating the role of the 936 group pangenome as a reservoir of genetic variation. The observed substitution rate homogeneity conforms to the neutral theory of evolution; hence, the neutral theory can be applied to phage genome evolution and also to genetic variation brought about by recombination.

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., (Copyright © 2018 American Society for Microbiology.)

In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Bacteriophage infection of lactic acid bacteria (LAB) is one of the most significant causes of inconsistencies in the manufacture of fermented foods, affecting production schedules and organoleptic properties of the final product. Consequently, LAB phages, and particularly those infecting Lactococcus lactis, have been the focus of intensive research efforts. During the past decade, multidisciplinary scientific approaches have uncovered molecular details on the exquisite process of how a lactococcal phage recognises and binds to its host. Such approaches have incorporated genomic/molecular analyses and their partnership with phage structural analysis and host cell wall biochemical studies are discussed in this review, which will also provide our views on future directions of this research field., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.

Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from ~1.1 x 105 to ~1.1 x 101 phage genomes per reaction, which corresponds to ~9 x 107 to ~9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother-bulk-cheese vat system. High levels of 936 and P335 phages were detected in the mother culture and the bulk starter, but also in the whey samples. Low levels of phages were detected in the cheese milk samples.

The protective effect of whey proteins on phages of lactic acid bacteria during heat treatment limits the recycling of whey proteins into cheese. To investigate this protective effect, we used lactoferrin (LF) as a whey protein model as a result of its unique physicochemical properties and its antiviral activity. First, the thermal inactivation of lactococcal thermoresistant virulent phage P1532 was measured in LF at 95 °C and under different pH values. Phage inactivation results revealed a strong protective effect of LF on P1532 phage at pH 5 but none at pH 7. The structural conformational changes of LF were then monitored by Fourier transform infrared and circular dichroism spectroscopies. Spectroscopic analysis showed that LF was unfolded after heating at pH 7, while it preserved its tertiary and secondary structures when heated at pH 5. There is a direct correlation between the thermal stability of LF and its ability to protect P1532 phage from heat treatment.

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. IMPORTANCE Streptococcus 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., (Copyright © 2017 Szymczak et al.)

Background: Despite continuous research efforts, bacterio(phages) infecting Lactococcus lactis starter strains persist as a major threat to dairy fermentations. The lactococcal P335 phages, which are currently classified into four sub-groups (I-IV), are the second most frequently isolated phage group in an industrial dairy context., Results: The current work describes the isolation and comparative genomic analysis of 17 novel P335 group phages. Detailed analysis of the genomic region of P335 phages encoding the so-called "baseplate", which includes the receptor binding protein (RBP) was combined with a functional characterization of the RBP of sub-group III and IV phages. Additionally, calcium-dependence assays revealed a specific requirement for calcium by sub-group IV phages while host range analysis highlighted a higher number of strains with CWPS type A (11 of 39 strains) are infected by the P335 phages assessed in this study than those with a C (five strains), B (three of 39 strains) or unknown (one of 39 strains) CWPS type., Conclusions: These analyses revealed significant divergence among RBP sequences, apparently reflecting their unique interactions with the host and particularly for strains with a type A CWPS. The implications of the genomic architecture of lactococcal P335 phages on serving as a general model for Siphoviridae phages are discussed.

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties., Competing Interests: Compliance with ethical standard Conflict of interest All the authors declare that he/she has no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

The lysogenic phage PLgT-1 is highly prevalent in Lactococcus garvieae, which is a serious bacterial pathogen in marine fish. Therefore, information regarding this phage is one of the key factors to predict the evolution of this bacterium. However, many properties of this phage, its complete genome sequence, and its relationship with other viral communities has not been investigated to date. Here, we demonstrated that the phage PLgT-1 was not only induced by an induction agent (Mitomycin C), but could be released frequently during cell division in a nutrient-rich environment or in natural seawater. Integration of PLgT-1 into non-lysogenic bacteria via transduction changed the genotype, resulting in the diversification of L. garvieae. The complete DNA sequence of PLgT-1 was also determined. This phage has a dsDNA genome of 40,273bp with 66 open reading frames (ORFs). Of these, the biological functions of 24 ORFs could be predicted but those of 42 ORFs are unknown. Thus, PLgT-1 is a novel phage with several novel proteins encoded in its genome. The strict MegaBLAST search program for the PLgT-1 genome revealed that this phage had no similarities with other previously investigated phages specific to L. garvieae (WP-2 and GE1). Notably, PLgT-1 was relatively homologous with several phages of Lactococcus lactis and 17 of the 24 predicted proteins encoded in PLgT-1 were homologous with the deduced proteins of various phages from these dairy bacteria. Comparative genome analysis revealed that the L. garvieae phage PLgT-1 was most closely related to the L. lactis phage TP712. However, they differed from each other in genome size and gene arrangement. The results obtained in this study suggest that the lysogenic phage PLgT-1 is a new member of the family Siphoviridae and has been involved in horizontal gene exchange with microbial communities, especially with L. lactis and its phages., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lactococcus lactis is an industrial starter culture used for the production of fermented dairy products. Pip (phage infection protein) bacteriophage-insensitive mutant (BIM) L. lactis DGCC11032 was isolated following challenge of parental strain DGCC7271 with C2viruses. Over a period of industrial use, phages infecting DGCC11032 were isolated from industrial whey samples and identified as C2viruses. Although Pip is reported to be the receptor for many C2viruses including species type phage c2, a similar cell-membrane-associated protein, YjaE, was recently reported as the receptor for C2virus bIL67. Characterization of DGCC7271 BIMs following challenge with phage capable of infecting DGCC11032 identified mutations in yjaE, confirming YjaE to be necessary for infection. DGCC7271 YjaE mutants remained sensitive to the phages used to generate pip variant DGCC11032, indicating a distinction in host phage determinants. We will refer to C2viruses requiring Pip as c2-type andC2viruses that require YjaE as bIL67-type. Genomic comparisons of two c2-type phages unable to infect pip mutant DGCC11032 and four bIL67-type phages isolated on DGCC11032 confirmed the segregation of each group based on resemblance to prototypical phages c2 and bIL67, respectively. The distinguishing feature is linked to three contiguous late-expressed genes: l14-15-16 (c2) and ORF34-35-36 (bIL67). Phage recombinants in which the c2-like l14-15-16 homologue gene set was exchanged with corresponding bIL67 genes ORF34-35-36 were capable of infecting a pip mutated host. Together, these results correlate the phage genes corresponding to l14-15-16 (c2) and ORF34-35-36 (bIL67) to host lactococcal phage determinants Pip and YjaE, respectively.

Lactococcus lactis is a starter bacterium commonly used in cheese making where it has an important role in acid-mediated curd formation as well as the development of flavour compounds. Industrial L. lactis strains can harbour one or more inducible prophages which when induced can affect cell growth and possibly lead to cell lysis. This is undesirable during growth and fermentation, but can beneficially lead to faster release of enzymes during cheese ripening. Lactococci can encounter multiple stress inducing conditions during the production of cheese, such as low and high temperatures, low pH, high osmotic pressure and long-term incubation. In this study, we tested the effect of these industrial stressors on prophage induction in two cheese making L. lactis subsp. cremoris strains (ASCC890049 and ASCC890310) as well as the laboratory strain L. lactis MG1363. Firstly, in order to identify inducible prophages in these strains we exposed them to the prophage inducing chemical mitomycin C (MMC) for 1 and 2h and then subjected the total genomic DNA to next-generation Illumina sequencing. Mapping of sequence reads back to the genome sequences revealed regions which contained a much higher fold coverage indicating DNA replication. These regions were amplified by up to 332-fold per cell (relative to the control tufA gene) and were identified as having similarities to different subgroups of P335 phages including MG-5, TP901-1, ul36.k1, bIL286, TP712 and BK5-T. Next, quantitative PCR was used to confirm the strong induction of prophages by MMC and then determine the copy number of the inducible prophages following exposure to various growth inhibitory levels of HCl, lactic acid, high temperature, NaCl, hydrogen peroxide and bacitracin. With the exception of a slight induction (2 to 4-fold) with hydrogen peroxide and long-term incubation after 21days in one industrial strain, none of the other stressors induced prophage DNA replication. These findings show that the repression system that maintains prophages in the dormant state in cheese making lactococcal strains is very tight and that several stressors encountered singularly are not predicted to be major inducers of prophage activation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: The Gram-positive bacterium Lactococcus lactis, used for the production of cheeses and other fermented dairy products, falls victim frequently to fortuitous infection by tailed phages. The accompanying risk of dairy fermentation failures in industrial facilities has prompted in-depth investigations of these phages. Lactococcal phage Tuc2009 possesses extensive genomic homology to phage TP901-1. However, striking differences in the baseplate-encoding genes stimulated our interest in solving the structure of this host's adhesion device. We report here the X-ray structures of phage Tuc2009 receptor binding protein (RBP) and of a "tripod" assembly of three baseplate components, BppU, BppA, and BppL (the RBP). These structures made it possible to generate a realistic atomic model of the complete Tuc2009 baseplate that consists of an 84-protein complex: 18 BppU, 12 BppA, and 54 BppL proteins. The RBP head domain possesses a different fold than those of phages p2, TP901-1, and 1358, while the so-called "stem" and "neck" domains share structural features with their equivalents in phage TP901-1. The BppA module interacts strongly with the BppU N-terminal domain. Unlike other characterized lactococcal phages, Tuc2009 baseplate harbors two different carbohydrate recognition sites: one in the bona fide RBP head domain and the other in BppA. These findings represent a major step forward in deciphering the molecular mechanism by which Tuc2009 recognizes its saccharidic receptor(s) on its host., Importance: Understanding how siphophages infect Lactococcus lactis is of commercial importance as they cause milk fermentation failures in the dairy industry. In addition, such knowledge is crucial in a general sense in order to understand how viruses recognize their host through protein-glycan interactions. We report here the lactococcal phage Tuc2009 receptor binding protein (RBP) structure as well as that of its baseplate. The RBP head domain has a different fold than those of phages p2, TP901-1, and 1358, while the so-called "stem" and "neck" share the fold characteristics also found in the equivalent baseplate proteins of phage TP901-1. The baseplate structure contains, in contrast to other characterized lactococcal phages, two different carbohydrate binding modules that may bind different motifs of the host's surface polysaccharide., (Copyright © 2016 Legrand et al.)

Model undefined thermophilic starter cultures were produced from raw milk of nine pasta-filata cheesemaking plants using a selective procedure based on pasteurization and incubation at high temperature with the objective of studying the microbial community dynamics and the variability in performances under repeated (7-13) reproduction cycles with backslopping. The traditional culture-dependent approach, based on random isolation and molecular characterization of isolates was coupled to the determination of pH and the evaluation of the ability to produce acid and fermentation metabolites. Moreover, a culture-independent approach based on amplicon-targeted next-generation sequencing was employed. The microbial diversity was evaluated by 16S rRNA gene sequencing (V1-V3 regions), while the microdiversity of Streptococcus thermophilus populations was explored by using novel approach based on sequencing of partial amplicons of the phosphoserine phosphatase gene (serB). In addition, the occurrence of bacteriophages was evaluated by qPCR and by multiplex PCR. Although it was relatively easy to select for a community dominated by thermophilic lactic acid bacteria (LAB) within a single reproduction cycle, final pH, LAB populations and acid production activity fluctuated over reproduction cycles. Both culture-dependent and -independent methods showed that the cultures were dominated by either S. thermophilus or Lactobacillus delbrueckii subsp. lactis or by both species. Nevertheless, subdominant mesophilic species, including lactococci and spoilage organisms, persisted at low levels. A limited number of serB sequence types (ST) were present in S. thermophilus populations. L. delbrueckii and Lactococcus lactis bacteriophages were below the detection limit of the method used and high titres of cos type S. thermophilus bacteriophages were detected in only two cases. In one case a high titre of bacteriophages was concurrent with a S. thermophilus biotype shift in the culture. This study largely confirms previous data on the composition of undefined thermophilic starters used for the production of traditional cheeses in Italy but it is the first one to systematically address the dynamics of the cultures under a repeated reproduction regime with backslopping., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cheese production is a global biotechnological practice that is reliant on robust and technologically appropriate starter and adjunct starter cultures to acidify the milk and impart particular flavor and textural properties to specific cheeses. To this end, lactic acid bacteria, including Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus and Leuconostoc spp., are routinely employed. However, these bacteria are susceptible to infection by (bacterio)phages. Over the past decade in particular, significant advances have been achieved in defining the receptor molecules presented by lactococcal host bacteria and in the structural analysis of corresponding phage-encoded receptor-binding proteins. These lactococcal model systems are expanding toward understanding phage-host interactions of other LAB species. Ultimately, such scientific efforts will uncover the mechanistic (dis)similarities among these phages and define how these phages recognize and infect their hosts. This review presents the current status of the LAB-phage interactome, highlighting the most recent and significant developments in this active research field.

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

Lactococcus lactis is used extensively worldwide for the production of a variety of fermented dairy products. The ability of L. lactis to successfully grow and acidify milk has long been known to be reliant on a number of plasmid-encoded traits. The recent availability of low-cost, high-quality genome sequencing, and the quest for novel, technologically desirable characteristics, such as novel flavour development and increased stress tolerance, has led to a steady increase in the number of available lactococcal plasmid sequences. We will review both well-known and very recent discoveries regarding plasmid-encoded traits of biotechnological significance. The acquired lactococcal plasmid sequence information has in recent years progressed our understanding of the origin of lactococcal dairy starter cultures. Salient points on the acquisition and evolution of lactococcal plasmids will be discussed in this review, as well as prospects of finding novel plasmid-encoded functions., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)