Lactococcal Phage Research Articles

Phage Biodiversity in Artisanal Cheese Wheys Reflects the Complexity of the Fermentation Process.

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.

Molecular Structure of Lactoferrin Influences the Thermal Resistance of Lactococcal Phages.

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.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

ABSTRACTBacteriophages 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.

Genetic and functional characterisation of the lactococcal P335 phage-host interactions

BackgroundDespite 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.ResultsThe 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.ConclusionsThese 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.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1.

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

Whey powders are a rich source and excellent storage matrix for dairy bacteriophages

Thirteen whey powders and 5 whey powder formulations were screened for the presence of dairy bacteriophages using a representative set of 8 acid-producing Lactococcus lactis and 5 Streptococcus thermophilus, and 8 flavour-producing Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides strains. Lytic L. lactis phages were detected in all samples, while S. thermophilus and Leuconostoc phages were present in 50% or 40% of the samples, respectively. Maximal phage titers were 6×107 plaque-forming units (pfu)/g of whey powder for L. lactis phages, 1×107pfu/g for Leuconostoc phages and 1×105pfu/g for S. thermophilus phages. In total, 55 phages were isolated and characterized. Thirty one of the 33 lactococcal phages tested belonged to the wide-spread 936 phage group. In the course of this study, a PCR detection method for Leuconostoc phages (Ali et al., 2013) was adapted to new phage isolates. Furthermore, a remarkably high stability of phages in whey powder samples was documented during a long-term storage period of 4 years.

Clear Plaque Mutants of Lactococcal Phage TP901-1.

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.

Comparative genomics and functional analysis of the 936 group of lactococcal Siphoviridae phages.

Genome sequencing and comparative analysis of bacteriophage collections has greatly enhanced our understanding regarding their prevalence, phage-host interactions as well as the overall biodiversity of their genomes. This knowledge is very relevant to phages infecting Lactococcus lactis, since they constitute a significant risk factor for dairy fermentations. Of the eighty four lactococcal phage genomes currently available, fifty five belong to the so-called 936 group, the most prevalent of the ten currently recognized lactococcal phage groups. Here, we report the genetic characteristics of a new collection of 936 group phages. By combining these genomes to those sequenced previously we determined the core and variable elements of the 936 genome. Genomic variation occurs across the 936 phage genome, such as genetic elements that (i) lead to a +1 translational frameshift resulting in the formation of additional structures on the phage tail, (ii) specify a double neck passage structure, and (iii) encode packaging module-associated methylases. Hierarchical clustering of the gene complement of the 936 group phages and nucleotide alignments allowed grouping of the ninety 936 group phages into distinct clusters, which in general appear to correspond with their geographical origin.

The Atomic Structure of the Phage Tuc2009 Baseplate Tripod Suggests that Host Recognition Involves Two Different Carbohydrate Binding Modules.

ABSTRACTThe 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.

Lactococcus lactisphage TP901–1 as a model forSiphoviridaevirion assembly

Phages infecting Lactococcus lactis pose a serious threat to the dairy fermentation sector. Consequently, they are among the most thoroughly characterized Gram positive-infecting phages. The majority of lactococcal phages belong to the tailed family of phages named the Siphoviridae. The coliphage lambda and the Bacillus subtilis phage SPP1 have been the predominant comparators for emerging siphophages both genomically and structurally and both phages recognize a membrane protein receptor. In contrast, the lactococcal P335 group phage TP901-1 attaches to cell wall surface polysaccharides. It is a typical "lambdoid" siphophage possessing a long non-contractile tail and a genomic architecture reminiscent of lambda and SPP1 despite low or undetectable sequence homology in many of its encoded products, especially those involved in host recognition. A functional analysis of the structural components of TP901-1 was undertaken based on the characterization of a series of mutants in the region encoding the capsid and tail morphogenetic elements. Through this analysis, it was possible to deduce that, despite the lack of sequence homology, the overall genomic architecture of Siphoviridae phages typified by functional synteny is conserved. Furthermore, a model of the TP901-1 assembly pathway was developed with potential implications for many tailed phages.

Carrier systems for bacteriophages to supplement food systems: Encapsulation and controlled release to modulate the human gut microbiota

Abstract The use of phages in food systems to modulate the human gut microbiota is currently gaining increasing interest. However, several studies have reported low viability of phages under gastrointestinal conditions. Furthermore, processed foods nowadays contain only few phage particles. Hence, this study aimed at encapsulating phages to increase their survival under gastrointestinal conditions allowing the release of phages in active form in the intestine. Lactococcus phage P008 was entrapped in different matrix materials, using three different encapsulation techniques (i.e., emulsion or extrusion method). Results showed that non-encapsulated phages were sensitive to acid conditions at pH 2.0. However, the investigated capsules which contained milk proteins provided good protection of phages at pH 2.0: a phage reduction of only 0.5 log-units was observed after incubation for 120 min at 37 °C. The release of phages from capsules after 2 h incubation in simulated intestinal fluid (pH 6.8) was also determined. Depending on the encapsulation technique, phages were completely released from the capsules. Micrographs confirmed these observations and showed a dissolution of the capsule matrix due to swelling and permeation of pancreatic enzyme. The study indicates that encapsulated phages may be targeted via food formulation to the intestine to modulate the human gut microbiota.

Bacteriophage module reshuffling results in adaptive host range as exemplified by the baseplate model of listerial phage A118

Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition.

Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH

The incorporation of whey protein concentrates (WPC) into cheese is a risky process due to the potential contamination with thermo-resistant phages of lactic acid bacteria (LAB). Furthermore, whey proteins can protect phages during heat treatment, thereby increasing the above risk. The main objective of this work was to understand this protective effect in order to better control LAB phages and maximize whey recycling in the cheese industry. First, the inactivation of a previously characterized thermo-resistant lactococcal virulent phage (P1532) was investigated at 95°C in WPC, in individual whey components β-lactoglobulin, α-lactalbumin, and bovine serum albumin as well as under different heat and pH conditions. The structural changes of the tested proteins were also monitored by transmission FTIR spectroscopy. Phage inactivation results indicated that the protective effect of whey proteins was pH and time dependent at 95°C and was not restricted to one component. FTIR spectra suggest that the protection is related to protein molecular structures and to the level of protein aggregates, which was more pronounced in acidic conditions. Moreover, the molecular structure of the three proteins tested was differently influenced by pH and the duration of the heat treatment. This work confirms the protective effect of WPC on phages during heat treatment and offers the first hint to explain such phenomenon. Finding the appropriate treatment of WPC to reduce the phage risk is one of the keys to improving the cheese manufacturing process.

Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ.

The lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage of antiQ by ABIQ was characterized using 5' rapid amplification of cDNA ends PCR and was located in an adenine-rich region of antiQ. We next generated a series of derivatives with point mutations within antiQ or with various numbers of antiQ repetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number of antiQ repeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile of antiQ. Interestingly, a point mutation in the putative pseudoknot structure of antiQ mutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.

Lactococcal 949 group phages recognize a carbohydrate receptor on the host cell surface.

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.

Investigating the requirement for calcium during lactococcal phage infection

Calcium is widely used in the study and successful propagation of virulent bacteriophages infecting lactic acid bacteria; however, it has not been assessed if and why this divalent cation is required for the infection process. Lactococcal phages are a persistent problem in the calcium-rich dairy environment and therefore were used as the model for this study. Using representative members of nine of the currently recognized ten lactococcal phage groups, encompassing phages of the Podoviridae and Siphoviridae families, we present data to suggest that calcium is not an explicit requirement for many of these phages. However, calcium expedited the pace of the lytic cycle for certain phages. Additionally, for calcium-dependent phages belonging to the 936 siphogroup, we could substitute this cation with magnesium or manganese, indicating that these phages are more adaptable than lactococcal phages of other groups. We postulate that the ability of phages to adapt to their environment and to harness the available mineral content may ultimately decide the success of a given phage infection. This may explain, in part, why 936 phages are one of the most frequently isolated phages in the dairy industry.

Microbial Population Dynamics in Presence of Lactococcal Bacteriophage During Ripening of Traditional Raw Milk Romanian Cheese

Abstract A massive Lactococcus lactis ssp.lactis c2 and e72 phage contamination of raw milk burduf cheese was revealed in all analyzed samples. Contamination with c2 phage specific for Lactococcus lactis ssp.lactis C2 is lower than the contamination induced by e72 phage. The contamination level with E.coli and coliforms in the core of burduf cheese was initially high, while the initial Staphylococcus spp. contamination was not high enough to pose a threat through toxin production. During the ripening period of the analyzed cheese, there was a high statistical significant decrease in E. coli, coliforms and staphylococci counts. This decrease was highly unexpected according to the scientific literature that indicates low lactococcal counts and low microbiological quality in bacteriophage contamination cases. The presence of wild bacteriophages, known to reduce the number of lactic acid bacteria, preventing the inhibition effect of the latter on contaminating bacteria growth, was proven not to induce a poor hygiene level of the raw milk burduf cheese, even if the initial contamination was considered to be high. This study is an example of natural, persistent and statistical significant lactococcal resistance to contaminating bacteriophages. The results are also scientifically significant due to the atypical time resistance of the microbiological balance between the contaminating bacteriophages and cheese bacterial microflora with positive consequences over cheese quality.

Application of a membrane technology to remove bacteriophages from whey

Whey from former cheese batches can be recycled either to increase the yield or to improve texture properties of fat reduced cheeses. However, in the case of the presence of bacteriophages, pasteurization may not be sufficient to eliminate phages in whey. Therefore, in this work, a cross-flow membrane filtration process was designed to separate whey proteins from whey-derived phages. Filtration experiments were carried out using native whey as model filtration medium, three polyethersulfone membranes (100, 300 and 500 kDa) that were studied in detail, and lactococcal phage P008. Filtration performance was characterized by phage retention, total whey protein permeation, and permeation of the major whey proteins α-lactalbumin and β-lactoglobulin. Filtration experiments showed that it is possible to reduce the number of phages in whey by filtration to a level at which subsequent phage multiplication is minimized and, concomitantly, high protein permeation through the membrane is ensured.

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

BackgroundSo-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.ResultsWhole 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.ConclusionsSMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-831) contains supplementary material, which is available to authorized users.

Cryo-electron microscopy structure of lactococcal siphophage 1358 virion.

Lactococcus lactis, a Gram(+) lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage. Here, we report the cryo-electron microscopy structure of the complete 1358 virion. Phage 1358 exhibits noteworthy features, such as a capsid with dextro handedness and protruding decorations on its capsid and tail. Observations of the baseplate of virion particles revealed at least two conformations, a closed and an open, activated form. Functional assays uncovered that the adsorption of phage 1358 to its host is Ca(2+) independent, but this cation is necessary to complete its lytic cycle. Taken together, our results provide the complete structural picture of a unique lactococcal phage and expand our knowledge on the complex baseplate of phages of the Siphoviridae family. Phages of Lactococcus lactis are investigated mainly because they are sources of milk fermentation failures in the dairy industry. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. In this study, we provide the cryo-electron microscopy reconstruction of a unique lactococcal phage that possesses genomic similarity to particular Listeria phages and has a host range restricted to only a minority of L. lactis strains. The capsid of phage 1358 displays the almost unique characteristic of being dextro handed. Its capsid and tail exhibit decorations that we assigned to nonspecific sugar binding modules. We observed the baseplate of 1358 in two conformations, a closed and an open form. We also found that the adsorption to its host, but not infection, is Ca(2+) independent. Overall, this study advances our understanding of the adhesion mechanisms of siphophages.