University of Groningen Lactococcus lactis bacteriophages: phage-host interaction and phage transduction Marcelli, Barbara

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Lactococcus lactis bacteriophages: phage-host interaction and phage transduction

Marcelli, Barbara

DOI:

10.33612/diss.118088081

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Publication date: 2020

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Marcelli, B. (2020). Lactococcus lactis bacteriophages: phage-host interaction and phage transduction. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.118088081

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Lactococcus lactis as an important starter culture in dairy fermentation processes

The Lactic Acid Bacteria (LAB) order comprises a big variety of genera, including Lactobacillus,

Lactococcus, Streptococcus, Leuconostoc, Pediococcus, Enterococcus, and Oenococcus (1) .

They are Gram-positive, non-sporulating, aerobic to facultative anaerobic rods and cocci and at present they are routinely utilized for the manufacturing of fermented foods such as dairy products, meats, vegetables, pickles, sauerkraut and alcoholic beverages (2).

Lactococcus lactis has been assigned the Generally Regarded As Safe (GRAS) designation

by the Food and Drug Administration (FDA) (3). It is the more predominantly used lactic acid bacterium in the dairy industry where it is employed as starter culture for the manufacturing of different products such as buttermilk, quark , and especially a wide variety of hard and soft cheeses (4). The most obvious contribution of L. lactis to the manufacture of dairy products is the fermentation of the lactose present in the starting food matrix with the consequent production of lactic acid that initiates the acidification of the milk (5). Lactococcal strains also greatly contribute to the flavor formation in dairy products, which consists of a combination of different compounds that can be either volatile or not volatile and results from the metabolism of either one or more of the main components of the milk: lipid, lactose and, to a greater extent, proteins (6–8). These molecules are generally transformed into the first flavor compound precursors via extracellular reactions performed by cell envelope-located enzymes. A second and more complex step follows and takes place inside the cell where the initial precursors are further introduced in different metabolic reactions. Some of the intermediate and end- products of this intricate net of metabolic pathways constitute the pool of aroma compounds typical of a given product (9–11). Another interesting feature that several L.

lactis strains possess is the ability to produce exopolysaccharides (12). It has been proven

that these extracellularly released sugars greatly contribute to some characteristics of dairy products like soft cheese, by acting as jellifying or emulsifier agents they can influence the texture of the end product giving, for example, a better mouth-feel (13). Besides from the dairy environment, L.lactis strains can be isolated from many different biological niches such as plants, fermented food and even the human vagina (14, 15). The adaptation to every single environment passes through acquiring new and essential metabolic skills needed to thrive in these changing surroundings and also through the loss of unnecessary functions (16). Lactococcal strains used in the dairy industry usually contain several plasmids that display a different G+C content compared to that of their chromosomal DNA, suggesting that they have been acquired more recently during strain evolution (17). Many of these plasmids encode properties that are of great relevance for the food manufacturing process; examples are lactose utilization, citrate metabolism, casein breakdown, bacteriophage resistance, bacteriocin production, and exopolysaccharide production (18–21).

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When L. lactis strains (and lactic acid bacteria in general) were initially employed for food processing, little or no information was available about their metabolic abilities. The first improvement of the protocols regarding the use of starter cultures has been the so called “back slopping” method. In this case, a small portion of the previously successful fermentation is inoculated into the new raw material in an attempt to guarantee the success of the new procedure, and to provide the bacterial population with the possibility to spontaneously develop metabolic features and to adapt to the environment (22). In Western countries, where the consumer demands are continuously increasing, a further and more precise step was made by adopting the approach of directly inoculating the milk with a few known selected L.lactis strains (23).

Fast acidification, flavor formation and phage resistance are the primary important abilities that a starter culture mix should possess (24). However, since a single strain does not usually possess all the metabolic abilities required for the manufacture of a specific end product, domesticated strains are often combined with newly isolated ‘wild strains’ derived from different milk environments, in order to optimize the pool of quality of the end products (25, 26). It has been shown that these two kinds of strains can, in many cases, coexist in the same environment contributing to the acidification or aroma formation via their different metabolic pathways in a process known as proto-cooperation (10). Besides the obvious advantages arising from the use of defined starter cultures in terms of qualities of the final products, some drawbacks also exist since such domesticated strains display a higher susceptibility to bacteriophage infection and a lower adaptation ability to changing environments (23).

For all these reasons, dairy companies are constantly investigating, among others, two extremely important aspects that contribute to the outcome of the fermentation process: metabolic abilities of starter strains and bacteriophage-host interaction in fermentation processes (24, 27). Understanding starter stains’ characteristics and dynamics is essential for the optimization of the fermentation processes and for the quality improvement of the end- products. Furthermore, since consumers’ demands in terms of quality and organoleptic characteristics is constantly evolving, it is of vital importance that dairy companies are provided with new and reliable tools for the genetic improvement of

L .lactis stains used in starter culture preparations (24).

On the other hand, bacteriophage infection being one of the main causes of fermentation halts or even failure, lactococcal phages have been extensively investigated in the past three decades (28). A thorough investigation of their properties is necessary in order to get a better understanding of phage evolution and phage-host interaction to move towards to an ever more precise control and confinement of bacteriophages spreading through production lines.

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The GMO issue in the European Union and the existing tools for L. lactis strain improvement for the dairy industry

As stated in the Directive 2001/18/EC of the European parliament (29), a genetically modified organism (GMO) is

“ an organism with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination” Genetic modification occurs at least through the use of the following techniques (Annex I B):

“- recombinant nucleic acid techniques involving the formation of new combinations of genetic material by the insertion of nucleic acid molecules produced by whatever means outside an organism, into any virus, bacterial plasmid or other vector system and their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation;

- techniques involving the direct introduction into an organism of heritable material prepared outside the organism including injection, macroinjection and micro-encapsulation;

- cell fusion (including protoplast fusion) or hybridization techniques where live cells with new combinations of heritable genetic material are formed through the fusion of two or more cells by means of methods that do not occur naturally.

Techniques which are not considered to result in genetic modification, on the condition that they do not involve the use of recombinant nucleic acid molecules or genetically modified organisms made by techniques/methods other than those excluded by Annex I B:

- In vitro fertilisation,

- Natural processes such as: conjugation, transduction, transformation - Polyploidy induction. “

In the European Union, any type of product containing or deriving from a genetically modified organism and intended for food and feed purposes, cannot be placed on the market before passing through a thorough examination conducted by the European Food and Safety Agency (EFSA) that has the role to assess the risk that GMOs may present to the environment, human health and animal safety (30).

This strict regulation, coupled with the negative public opinion towards the consumption of GMO-derived products, places an important restriction on the type of technologies that food industries, and in particular dairy industries, can employ to improve bacterial strains used as starter cultures (31).

Recombinant DNA technology that is routinely used in research laboratories would be the perfect strategy for selective genotypic improvement of microorganisms especially

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due to its accuracy and reliability. However, this approach gives rise to organisms that would be considered as GMOs and would not obtain the EFSA authorization to be put on the market.

In this scenario, dairy industries have to overcome this problem by constantly identifying alternative methods for the improvement of LAB strains.

When trying to improve the characteristics of a starter culture strain, the aim of the genetic modification can vary: increasing the expression of a specific gene to obtain higher protein production, silencing of genes, the activity of which can interfere with the quality of the end product, or modulating the expression of one or more genes or introducing new genes (24).

Approaches that have been explored or that are nowadays used to serve all the above mentioned purposes are: (i) the use of food-grade transformation vectors, (ii) random mutagenesis carried out with agents such as UV light, (iii) adaptive mutation, the approach via which a strain is forced to grow in a certain growing condition compelling it to slowly adapt to the new environment via the development of new abilities, (iv) dominant selection, a method that focuses on the designing of a specific selection mechanism that only allows the growth of strains carrying the desired mutations.

With the exception of the use of transformation vectors, the remaining mentioned techniques do not pose any question on the food-grade denomination of the modified strain as they do not contemplate the transformation of the strain under analysis with any external genetic material, but are instead focused on the direct modification of the genotype of the strain itself. The obvious disadvantages of these techniques are, firstly, the inevitable need for screening a large array of different mutants in order to find the one that displays the wanted phenotype, and secondly, the risk to introduce unwanted mutations along with the intended ones (24).

Starting from the early nineties, a big effort has been put on the development of food grade transformable vectors (32, 33). However the laborious process of constructing such vectors poses a number of limitations that greatly restrict their use. The need to utilize a plasmid of which the origin of replication does not compete with the ones of the natural plasmids carried by the strain to be modified; the difficulty of finding food grade selectable markers whose expression can be detected in the receiving strain, and the obvious necessity for the plasmid to be transformed via naturally occurring methods, are among the major restrictions concerning this approach (34).

It is clear from this overview that the process of improving starter strains used for the

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production of fermented food is still quite long and tedious. Clearly, in an industrial setup this aspect of fermentation optimization is of crucial importance making it imperative to continuously develop new and reliable techniques to reach this goal.

The European legislation regarding the use of GMOs in the food industry has not changed in the past decades despite repeated requests from both industrial and scientific parties to re-evaluate some of the restrictions on the topic. For this reason naturally-occurring gene transfer methods such as conjugation, transformation, bacteriophage transduction, and nanotube formation have been taken into consideration in the past years with some promising results (35).

Bacteriophage replication and the role of phages in horizontal gene transfer

Bacteriophages are viruses that infect bacterial cells; with an estimated 1031 _particles

present in all type of environments that can be found in the biosphere, they are among the most abundant biological entities on the planet (36).

All known bacteriophages infecting L. lactis possess a dsDNA genome that is tightly packaged inside a proteinaceous head called capsid to which the complex tail structure involved in phage-host interaction is attached (37). As all bacteriophages, they can undergo two main replication cycles known as lytic and lysogenic (36, 38) (Figure 1).

Figure 1. Schematic representation of the life cycle of a bacteriophage.

Green and orange arrows indicate the lytic and lysogenic cycle, respectively. The different steps of both cycles are indicated by corresponding color-coded numbers.

The first is typical of bacteriophages known as strictly lytic: in this case, the initial step of the infection is the recognition and binding, mediated by the phage receptor binding protein (RBP), of the host receptor present on the surface of the bacterial cell. Successful

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binding triggers the injection of the viral genome into the host cytoplasm where phage multiplication takes place: the bacterial replication, transcription and translation machineries are hijacked to synthesize new intact virions. First, the viral DNA circularizes to be replicated in the form of concatamers composed of multiple copies of phage genomes from which viral proteins are synthesized. Once the structural proteins composing the head (capsid) and tail structure of the virion are produced, the viral-encoded enzyme terminase inserts one copy of the phage genome inside each empty capsid via an ATP-dependent mechanism. When the genome has been packaged inside the capsid, a tail is attached to the latter to form an intact viral particle. The last step of the infectious cycle is the burst of the newly formed viral progeny from the host cell: the bacterial cell wall is degraded from within by the concerted action of the viral enzymes holin and endolysin, allowing the release of the virions into the environment.

The lysogenic life cycle is, instead, typical of the temperate bacteriophages. After the viral genome has been injected inside the host cytoplasm, these bacteriophages can take the decision of integrating their genetic material inside the host chromosome. When integrated, these phages are called prophages, and in this state they can be replicated together with the bacterial chromosome for many generations. External factors that impair the functional structure of the host chromosome, like UV or antibiotics exposure, can trigger the excision of the prophage which subsequently enters the lytic replication cycle.

Two less investigated bacteriophage life cycles known as pseudolysogeny and carrier state life cycle (CSLC) can, however, occur in bacteriophages and also in lactococcal ones (39, 40).

The first case describes the situation in which a temperate bacteriophage doesn’t commit to either a lysogenic or a lytic life cycle but, instead, is present in an episomic state inside the host cell where it can be replicated albeit at a lower frequency than in a classical lytic cycle. It has been hypothesized that a temperate bacteriophage enters pseudolysogeny in situations of bacterial starvation: when the host of the phage is not able to replicate at normal rate, pseudolysogeny guarantees survival of the phage under the harsh conditions. Normal bacteriophage replication is restored once host starvation is resolved (39). Although the hypothesis of this being a strategy adopted by the phages to survive suboptimal growing conditions seems intuitive, it has not been excluded that pseudolysogeny just represents a normal intermediate step in temperate bacteriophages replication (40).

The CSLC, undergone by lytic phages, depicts a situation of equilibrium between the bacterial and the phage populations. In this scenario the majority of the bacterial

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population is resistant to the phage, although a subpopulation of sensitive cells exists and allows the continuous production and release of intact virions (40). CSLC allows both the host and the phage to thrive and it has been described to also occur in situations of optimal growing conditions of the host (conversely to pseudolysogeny) and during exponential cell growth (40). The natural triggers of such replication cycle are still poorly understood, but advantages for the bacteria derived from the establishment of such a relationship, have been described (41, 42). A recent study conducted on mixed LAB cultures used in dairy production even showed that the presence of carrier cells allows the stabilization of the whole bacterial population and, consequently, a better adaptation of the starter culture to the dairy fermentation conditions and to attacks by external bacteriophages (23).

During active replication of phages, mistakes in the process of packaging of viral genomes inside empty capsids can lead to a phenomenon known as transduction. In this case, the phage terminase can erroneously insert host chromosomal or plasmid DNA inside an empty capsid giving rise to a virion, known as transducing particle, that can carry exclusively host DNA or a mixture of viral and bacterial DNA (43). Given that the tail structure of a transducing particle is identical to that of a normal virion, they retain the ability to infect a new host and to inject their genetic material, derived from the host in which they were initially formed. Upon injection, this bacterial DNA can integrate into the new host chromosome or can be retained as episome. Two types of transduction are known: generalized and specialized (40) (Figure 2). Generalized transduction entails the transfer of any given fragment of bacterial chromosomal or plasmid DNA and both lytic and temperate phages can perform it (40). Specialized transduction is typically performed by temperate phages: when the prophage excises from the host chromosome to enter lytic replication, a fragment of chromosomal DNA immediately adjacent to the phage genome can be erroneously excised with it and consequently packaged into an empty capsid to be subsequently transferred to a new host (40).

Phage transduction is one of the horizontal gene transfer (HGT) mechanisms that are known to take place in nature together with conjugation, transformation and nanotubes transfer (35). In HGT, genetic material is transferred among two bacteria of the same or of different species (44). Attempts have been made to exploit this natural phenomenon to mobilize desired genetic traits among different LAB used in dairy fermentations to enhance their metabolic activities (45–48). This approach, as it is based on naturally occurring mechanisms, would give rise to modified starter cultures that should receive approval for use in the foods to be marketed in the European Union.

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Figure 2. Schematic representation of bacteriophage transduction.

A: generalized transduction; B: specialized transduction.

The majority of successful attempts, in L. lactis, report on chromosomal or plasmid DNA transfer via phage transduction mediated by temperate bacteriophages, while very little if no information is available on the ability of strictly lytic bacteriophages to transfer chromosomal DNA. In Chapter 4 of this thesis a different approach based on phage-mediated HGT will be described where strictly lytic lactococcal bacteriophages are employed for the mobilization of genetic material among lactococcal strains.

Lactococcal bacteriophages in the dairy environment

The current taxonomic classification of lactococcal bacteriophages is maintained at the International Committee on Taxonomy of Viruses (ICTV) (https://talk.ictvonline.org/),

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and relies mainly on electron microscopic analysis, DNA-DNA hybridization, and genome sequence comparisons (49, 50). This approach, adopted for all known bacteriophages, has proven to be useful for the classification of bacteriophages so far. However, since metagenomics analytic approaches and analysis techniques became more accessible and are more routinely employed, the necessity of revising this classification method arise. The ongoing debate points out that a strategy that strongly relies on morphology-based metrics is consequently not taking into consideration all those bacteriophages for which only the genome sequence is known and have not yet been isolated, propagated and visualized. This leads to underestimation of the diversity and mosaicism that is typical of bacteriophage genomes. This, in turn, can lead to the establishment of taxonomical families composed of phages that are evolutionary very distant and that should be, instead, divided into different and more specific families or subfamilies (51, 52). For this reason, a more genomic-based approach for taxonomic classification of bacteriophages has been proposed and a significant change in current virus taxonomy, is foreseen to happen in the coming years (53).

Although improvement of bacteriophage classification is actively being pursued, at present the scientific community is still heavily relying on the original taxonomical system. In this context, bacteriophages infecting L. lactis are classified as belonging to the Caudovirales order as they all possess a dsDNA genome (54). They are further divided in either the Siphoviridae (with long-non contractile tail, and isometric or prolate capsid) or the Podoviridae (with short non-contractile tail and icosahedral capsid) families, with the Siphoviridae phages being the more abundant ones (55).

The most recent partitioning of lactococcal phages separates them into ten different species, namely 936, c2, P335, KSY1, Q45, 1358, P087, 949, P034 and 1706 (Figure 3). The members of the first three species are the most commonly isolated in dairy plants (56, 57). Bacteriophages of the 936 and c2 species are strictly lytic and have highly conserved genomes both at the organizational and nucleotide sequence level (58, 59). The 936 phages have been proven to be able to acquire different genes or gene modules, especially in the regions of the genome that specify morphogenesis and packaging functions (60). This allows them to quickly adapt to the host receptors and defense mechanisms. Together with their ability to overcome thermal and biocidal treatments, it explains why these are still possibly the lactococcal phages that cause the highest starter culture infection events in dairy industry (61).

Bacteriophages of the c2 species possess some of the smallest genomes among the lactococcal phages (about 21 kb) (58). Given the relative ease with which c2 bacteriophage insensitive mutants (BIMs) can be isolated, and because phage resistance mutations in these strains do not affect their fermentation abilities, the incidence of c2 bacteriophages in dairy environments seems to have been diminished in recent times (62).

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Figure 3. L. lactis phage classification.

Electron micrographs of a representative of each phage species. The species names are given under the corresponding images. Phages are divided into two families: Siphoviridae and Podoviridae, as indicated at the top of the figure. Adapted from (56).

The P335 species, on the other hand, is more heterogeneous as it comprises both lytic and lysogenic phages that are genetically more distantly related among each other. Members of this species show a very high genome plasticity due to their high ability to acquire single genes or functional gene clusters (63). Being a very heterogeneous group, no single gene has been identified to be present in all the P335 phages described so far (64, 65). Bacteriophages have been assigned to this species on the basis of Southern hybridization and morphological analysis. A more recent classification identified five different subgroups based on the presence of homologous functional regions and baseplate component structure. The five subgroups are the following: P335/ul36/Tuc2009/TP901-1, BK5-T/4268, r1t/LC3, and Q33/BM13 (63).

Members of the remaining lactococcal bacteriophage species are more rarely isolated from failed fermented processes and some species are represented by only one isolate (57). Some of their structural (e.g. long tail) and replication characteristics (e.g. long latent period) pose an impediment to their proliferation under harsh fermentation conditions like mechanical constraints, high temperature and fast production rate that are typical of dairy productions and sterilization protocols (66, 67). Nonetheless, these

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phages can be isolated from failed cheese productions and several studies have shown how they can evolve both through homologous recombination among members of more common lactococcal phage species (68) and via gene acquisition from phages infecting bacterial species more distantly related to L. lactis (66). For this reason, their role and adaptation to the dairy environment should not be neglected.

Lactococcal phage host recognition

Infection of dairy starter cultures by bacteriophages can lead to fermentation failure and consequent important economic losses for the dairy industry (69). For this reason, in the past two decades, a big effort has been put into the characterization of lactococcal bacteriophages to gather information on the way they interact with and proliferate in a bacterial population (70). With this information in hand, dairy companies design specific starter culture rotation plans in which bacterial cultures with comparable metabolic activities but different phage sensitivities are used in rotation in order to lower the chances for bacteriophages spreading within a production plant (62). To control, predict and understand phage proliferation in dairy environments, food companies need to be provided with both fast screening methods to identify the type of phage causing a starter culture infection, and information regarding the way these phages recognize their hosts and proliferate within a bacterial population.

Multiplex PCR approaches have been developed to identify the species to which the bacteriophage(s) belong that cause a starter culture infection (71, 72). Three different primer pairs have been specifically designed to recognize genes encoding conserved (usually structural) proteins, exclusively present in every phage of the three most common species 936, c2 and P335. The three primer pairs are used together in a single PCR reaction performed on a sample of the failed fermentation process and quickly provide an initial screening of the phage population present in the sample. Some difficulties have been reported in the optimization of this type of multiplex PCR analysis because of the absence of a unique conserved gene in all the members of the P335 species. This creates the risk of overlooking, in a given sample, of P335 phages (65). However, the technique represents a valuable source of first-hand information and it is currently routinely employed in industrial settings.

Initial studies on phage-host interaction mechanisms in dairy plants of 936, c2 and P335 species focused on a few selected bacteriophages that were used as references for each species. Bacteriophages sk1, jj50 and p2 belonging to the 936 species (73), the eponymous phage c2 (58), and the P335 phages Tuc2009 and TP901.1 (74, 75) have been thoroughly investigated at the genomic, structural and phenotypical level. Years of investigations of lactococcal phages have produced a large amount of information regarding the type of receptors recognized by these phages, as well as their general genome organization and the structural details of receptor binding.

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It is now commonly known that phages belonging to the 936 and P335 species recognize saccharidic receptors (76, 77). These molecules form part of the cell wall polysaccharide pellicle (CWP) that surrounds lactococcal cells and that is encoded by a specific operon. Three major types of CWP-encoding operons have been identified so far in L. lactis namely type A, B, and C. Each of these contains a conserved region of which the gene composition and nucleotide sequence are nearly identical in all the three operons, and a second, less conserved region, that is typical for each of the three operon types (78). The CWP encoded by each operon differs in the relative ratio of the four sugars composing the pellicle: glucose, galactose, glucosamine and rhamnose. The specific sugar composition of the CWP of a few lactococcal strains has been already elucidated, broadening the degree of detailed information available on phage-host interaction (76, 77, 79–81).

Initial studies on bacteriophage c2 showed that this phage, in contrast to the species mentioned above, recognizes a proteinaceous receptor in a two-step process in which the phage first reversibly binds a sugar molecule on the surface of the cell, and only later irreversibly attaches to a protein (82). The Pip protein (Phage Infection Protein) was thought, for many years, to be the only receptor recognized by c2 bacteriophages (83). During the last two decades the possibility to sequence large numbers of bacterial and bacteriophage genomes gradually increased until the point that, nowadays, genome sequencing is routinely used in many laboratories, greatly increasing current knowledge on lactococcal bacteriophages. Studies on c2 phages isolated from dairy production lines and their BIMs revealed that phages belonging to this group can, in fact, recognize a second protein called YjaE as host receptor (84). In silico analysis combined with phenotypic results allowed classifying c2 bacteriophages into two separate eponymous subgroups c2 and bIL67. A correlation between the nucleotide sequence of three viral structural genes involved in host recognition and the corresponding protein that the phages recognize, could be made revealing that phages of the c2 subgroup recognize Pip as receptor while members of the bIL67 group recognize YjaE (84, 85).

Following a similar approach of combining in silico and in vivo approaches on a large group of 936 bacteriophages allowed connecting the RBP sequences of these phages with the CWP type in which their host receptor is embedded (60, 78). Similarly, a comparative genomic analysis on 17 phages belonging to the P335 species revealed the high degree of structural differences among the RBPs of these isolates and, concomitantly, their possible preference for receptors embedded in CWP of the type A (86).

Bacteriophages that belong to species less commonly isolated in dairy environments have received less attention from the scientific community. Nonetheless, members of these rare species can be isolated from failed fermentation samples and can contribute to halts in production lines, thus making them a threat that cannot be ignored. Given

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the little information available on rare lactococcal phages, a number of separate studies have aimed at reducing this knowledge gap and have analyzed the nature of the receptors recognized by members of different lactococcal phage species, such as 949, 1358 and 1706. These studies have revealed that sugars composing the CWP of the lactococcal cells are, also in these cases, used as host receptors (66, 87, Chapter 3, this thesis).

Analysis of big data obtained by next generation sequencing (NGS), together with new and improved biotechnology techniques, should enable the dairy industry to rely on detailed information in order to improve existing fermentation processes or to engineer new ones. It is obvious that a continuous influx of data on both widespread and rarer bacteriophages, and the proper analysis of the embedded information is of vital importance in this respect.

Research rationale and thesis outline

This thesis work aimed at broadening the current knowledge on bacteriophages infecting the Gram-positive lactic acid bacterium Lactococcus lactis. Being one of the main causes of fermentation failure in the dairy industry, lactococcal bacteriophages have received great attention by the scientific community in the past four decades and they are, at present, the most investigated bacteriophages after those infecting the Gram-negative bacterium

Escherichia coli. Dairy companies that routinely employ L. lactis in starter culture

preparations, greatly rely on the continuous increase in the knowhow on lactococcal bacteriophage-host interaction, host receptor recognition, phage adaptation to new hosts and environments, and phage genome structure and composition. Besides delivering valuable information that can be exploited to improve or protect food fermentation processes, research on lactococcal bacteriophages is of great importance as it can provide reference information for research on bacteriophages infecting Gram-positive bacteria. In Chapter 2 we analyze a lactococcal bacteriophage collection with the aim of identifying characteristics of the phage isolates that are of interest for the dairy industry. The phage library is composed of 22 specimens isolated from failed fermentation processes from factories located at different places in the world. Their complete genome sequences were determined, they were assigned to specific species assigned, and their host range was tested against a panel of 18 lactococcal dairy-derivatives and laboratory strains. Particular attention was given to the bacteriophages’ genetic determinants for host recognition. Phenotypic and in silico analysis method were combined to assess whether the current knowledge on phage-host interaction also applies to our collection of bacteriophages and bacterial strains. This approach allowed, on the one hand, contributing to the growth of genome sequencing data on lactococcal bacteriophages, and, on the other hand, identifying at least one exception regarding the interaction of c2 bacteriophages with their hosts. Furthermore, we were able to identify a new and yet uncharacterized subgroup of bacteriophages of the 936 species.

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The sequencing data derived from this-large scale analysis led to the discovery of a new member of the rare lactococcal bacteriophage species 1706, named CHPC971. In Chapter

3 we take a closer look into this new isolate, aiming at identify the nature of the host

receptor recognized by this phage. Combining competition assays, microscopy data and

in silico analyses we obtained strong evidence that phage CHPC971 recognizes a sugar

receptor embedded in the cell wall polysaccharide pellicle of its host, and that rhamnose plays an important role in this interaction. Finally, a detailed analysis of the phage genome enabled identifying gene candidates involved in host recognition by phage CHPC971 and, possibly, by other members of this yet poorly characterized lactococcal phage species.

Chapter 4 addresses the possible exploitation of bacteriophage-mediated lateral gene

transfer (bacteriophage transduction), as a tool for genome alterations in L. lactis starter cultures. Such a technique would deliver bacterial strains that should be considered as non-GMO and, as such, should be allowed for use in the food market in the European Union. Since information regarding the use of temperate bacteriophages for the transfer of industrially relevant plasmids among lactococcal strains is already available in literature, we decided to explore the possibility of employing strictly virulent bacteriophages for this purpose. Three phage isolates belonging to the c2 species were selected and tested for their ability to transfer chromosomal and plasmid DNA among lactococcal strains. A transposon-based library constructed in the laboratory model strain L. lactis MG1363 and two different plasmids were employed, respectively, to test the two possibilities. Plasmid DNA was transferred successfully with all the tested bacteriophages, and this data provides valuable information for setting up of an efficient phage transduction procedure using lytic phages.

In Chapter 5 a less commonly investigated phage-host interaction mechanism, known as the carrier state life cycle (CSLC), is investigated in L. lactis. Our data provides evidence for the establishment of CSLC in the laboratory strain L. lactis MG1363 by a c2 bacteriophage. Adopting a combined approach of phenotypic and in silico analysis allowed demonstrating that the interaction between the newly identified bacteriophage with a second c2 bacteriophage infecting L. lactis MG1363 leads to the release of a mixed phage population, the members of which differ with respect to their genome sequence and host range. We propose that this is one of the evolutionary mechanisms that allow bacteriophages to quickly adapt to different hosts and environments. These results are valuable not only for preventing bacteriophage infection of industrial starter strains, but for any experimental approach that exploits bacteriophages such as, for example, used in phage therapy.

The most important results obtained in this thesis work are summarized and further discussed in Chapter 6.

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