**6. Defense mechanisms of lactic acid bacteria**

It is well documented that lactic acid bacteria evolved defense systems against bacteriophages, which allow them to survive in an environment full of their predators. These anti-phage systems have been organized into five groups depending on the manner by which they operate: (i) inhibition of phage adsorption, (ii) blocking of phage DNA injection, (iii) restriction modification systems, (iv) phage abortive infection systems, and finally, the most recently described, (v) CRISPR/*cas* systems. The knowledge about natural phage resistance mechanisms together with a set of genetic tools were applied to develop also (vi) engineered defense systems that confer higher levels of resistance and/or broader phage specificity.

### **6.1. Inhibition of phage adsorption**

26 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

morphology.

world cannot be underestimated.

The majority of known viruses are bacteriophages, which infect cells of Eubacteria and Archaea. It is also accepted that most phages (96%) isolated so far belong to one taxonomic order of *Caudovirales* [9]. Bacteriophages within this order contain tails and a linear dsDNA genome. They are further classified into three phylogenetically linked families of: *Myoviridae, Siphoviridae, Podoviridae* [9]. *Myoviridae* phages contain a long and contractile tail, while *Siphoviridae* and *Podoviridae* are equipped with a non-contractile tail, long and short, respectively [10]. Isometric heads are dominating (85%) in the morphology of phages from all three families [11]. It is worth to mention that 61% of known phages are classified into the *Siphoviridae* family, of which most of them infect strains of enterobacteria (906 phages), *Lactococcus* (700), *Bacillus* (380) and *Streptococcus* (290) [9]. Apart from the tailed *Caudovirales* phages, there are others demonstrating filamentous, pleomorphic or polyhedral

Bacteriophages, although simple in organization, are the most diverse life forms in the biosphere. Their apparent heterogeneity is reflected by various features – both morphological as genetic, and their persistence on Earth, estimated as high as 1031, outnumbers by far their bacterial hosts [12]. Phages inhabit various niches, like oceans [13], thermal waters [14], gastrointestinal tract [15] and superficial ecosystems created by man, including fermentation tanks in dairy industry [16]. Hence, their impact on the microbial

Bacteriophage genome structure, indicating linear and double-stranded characteristics of the DNA molecule, containing or not cohesive ends and sometimes presenting terminal redundancy and circular permutation, describes the general feature of LAB phage genomes.

To enter the host, phages firstly come in contact and adsorb to the bacterial cell wall. The adsorption process has been well studied in Gram-negative bacteria, where it was found that two components are involved in the phage-host interaction. One of them is a receptor located in the bacterial cell envelope (membrane or wall), whereas the second component, called the receptor binding protein (RBP), is presented on the phage surface. RBP is responsible for recognition and binding of the phage particle to the bacterial receptor [17]. In the first stage of phage infection, the RBP protein recognizes and binds to a suitable sugar receptor. However, such binding is reversible and thus, the initial phage-bacteria interaction does not ensure commencement of a successful infection event. In contrast to this, in the second stage, a stable phage attachment to the bacterial cell occurs due to an irreversible binding between proteins located on bacterial and phage surfaces [18]. Both stages of adsorption are observed in Gram-positive bacteria: phages that attack *Lactococcus lactis* cells bind to specific receptors, mainly sugars, located in the cell wall. It is widely known that rhamnose, glucose, galactose, and galactosamine are compounds with which the phage RBP interacts at the initial stage of adsorption [19]. In the case of *Lactococcus* c2-type phages, effective infection requires interaction between phage and the bacterial protein Pip (phage infection protein) [20]. The Pip protein of *L. lactis* is an integral membrane protein [21] and

**4. Molecular mechanisms of phage infection of LAB** 

Basic mechanisms of inhibition of phage adsorption to the bacterial cell are associated either with physical masking of the receptor or with changes in its structure, or even with its

absence in the cell envelopes [24]. Lack of a functional receptor might be due to spontaneous mutations in the genetic material, leading in turn to bacteriophage insensitive mutants (BIM). A good illustration of the BIM phenomenon is a lactococcal mutant in the chromosomally-encoded *pip* gene. The resultant strain is unable to interact with phages of the c2 group, revealing high level of c2-specific resistance [24] (for further details on BIMs see section 12.2.).

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

hand, only few of them have been well characterized [28]. Therefore, the mechanism preventing entry of phage DNA to the cell is still poorly understood, both in LAB and other microorganisms. Surprisingly, it was discovered that most *sie* genes are located within the prophage regions of the bacterial chromosome [28]. However, the first lactococcal injection blocking system was identified on the pNP40 plasmid, which blocks DNA penetration specifically for φc2 phage of the lactococcal c2 phage group [29]. As it was described in the previous section, the membrane Pip protein is essential for c2 adsorption to *Lactococcus lactis*. It was speculated that the pNP40-encoded protein product might have an impact on the activity, production, or membrane insertion of Pip, thereby affect its biological function and prevent phage DNA entry [29]. The first description of a *sie* system of *Lactococcus* was published in 2002 and referred to the P335-type temperate lactococcal bacteriophage Tuc2009 [30]. After integration of the bacteriophage Tuc2009 genome into the lactococcal chromosome, the prophage protein Sie2009 is produced and blocks superinfecting phage DNA entry into the cell. The blocking mechanism has not been fully elucidated; nevertheless, it has been proposed that Sie2009 interacts with factor(s) responsible for initiating the phage DNA release from the capsid. Alternatively, the Sie2009 protein might interact with cell membrane proteins that are essential for DNA translocation. The effect of Sie2009 seems to be analogous to the effect of the lysogenic phage repressor (CI) preventing re-infection. In contrast, the presence of the *sie2009* gene determines resistance to various phages, also to phages from other species [28,30]. Similarly to lactococci, in lactobacilli prophages are also a common phenomenon [31]. Comparative genomics of lactobacilli revealed the presence of genes coding for putative proteins with a close sequence match to a surface-exposed lipoprotein encoded by bacteriophage TP-J34 of *Streptococcus thermophilus*, another bacterial species used in industrial milk fermentation processes. The TP-J34 prophage carries a Sie-like system consisting of the *ltp* (lipoprotein of temperate phage) gene, encoding a surface-exposed lipoprotein of biologically proven phage-resistance functions. In view of the fact that the *sie* genes of lactic acid bacteria are located on lysogeny modules of prophages and confer infection exclusion, they have been termed phage-derived

Following successful injection of DNA, phage infection might be completed or hindered by the presence of restriction modification systems (RM). RM systems comprise two activities represented by the following enzymes: endonuclease (restriction) and methyltransferase (modification) [33]. Simultaneously, both activities are specific to the same target sequences. The endonucleolytic activity is responsible for degradation of invading foreign DNA, including phage DNA, which lack a unique methylation pattern, while the methyltransferase activity protects the host DNA against degradation by introducing a methyl group into a specific nucleotide of the target site [34]. In detail, phage DNA usually reveal different methylation patterns than those recognized by innate RM systems. Unmethylated target sequences are significantly susceptible to endonucleolytic attack,

phage resistance systems [32].

**6.3. Restriction modification systems** 

to Lower Phage Contamination in Dairy Fermentation 29

Mechanisms preventing phage adsorption are not only mediated by the bacterial chromosome, but also by acquired plasmids. The best documented plasmid-encoded mechanisms of inhibition of phage adsorption rely on either direct synthesis of cell surface antigens or the production of extracellular carbohydrates. Of the two modes of action, the former reveals phage specificity, whereas the latter seems to restrict access to the bacterial cell for various harmful factors, including bacteriophages [25]. Studies carried by Tuncer and Akcelic demonstrated that a 28.5-kb plasmid, isolated from *L. lactis* subsp. *lactis* MPL56, causes complete inhibition of four lactococcal phages due to the production of a 55.4-kDa protein [25]. The protein exhibits similarity to lectins, a group of proteins that adsorb to specific monosaccharide components of polysaccharides in the cell wall, hence, impairing specific recognition of the phage receptor sites by these four phages. Thus, this plasmidencoded 55.4-kDa protein shields specifically the galactose-containing receptor rather than interacts with the phage, in other words, the bacterial lectin and the phage RBP compete for the receptor [25]. Another example of physical masking of the receptor is the plasmidmediated production of extracellular carbohydrates, called exopolysaccharides (EPS) [26]. Such EPS envelope coats the cell surface giving bacteria extra protection, not only against bacteriophages, but also against desiccation. There is some evidence that EPSs contain sugar residues that are similar or even identical to initial phage receptors. Therefore, phage insensitivity of LAB strains that carry EPS-encoding plasmids, for instance, pCI658, might be due to phage immobilization by binding to EPS [26]. On the other hand, polysaccharides have an impact on the properties of dairy products, like: texture, viscosity and smoothness of mouthfeel. Thereby, application of EPS-producing phage-resistant strains might be limited to a narrow range of dairy products [25-26].

### **6.2. Blocking of phage DNA injection**

After phage binding to the receptor, phage DNA is introduced into the bacterial cell. In the cytoplasm, phage genetic information is amplified and consequently progeny particles are produced. However, studies of Watanabe on the interaction between phage PL-1 and a *Lactobacillus casei* strain showed no bacterial lysis, despite phage adsorption to cell envelopes [27]. An electron microscopy image indicated that the phage DNA remains intact in the capsid. In contrast to this, a significant increase in the number of empty capsids was observed on the surface of the sensitive strain. In the light of this evidence, it is obvious that phage DNA injection might be interrupted, although the adsorption of phages to the cell surface occurred. Intensive attempts to elucidate the injection blocking phenomenon have allowed identifying different Sie (superinfection exclusion) or Sie-like systems. On the other hand, only few of them have been well characterized [28]. Therefore, the mechanism preventing entry of phage DNA to the cell is still poorly understood, both in LAB and other microorganisms. Surprisingly, it was discovered that most *sie* genes are located within the prophage regions of the bacterial chromosome [28]. However, the first lactococcal injection blocking system was identified on the pNP40 plasmid, which blocks DNA penetration specifically for φc2 phage of the lactococcal c2 phage group [29]. As it was described in the previous section, the membrane Pip protein is essential for c2 adsorption to *Lactococcus lactis*. It was speculated that the pNP40-encoded protein product might have an impact on the activity, production, or membrane insertion of Pip, thereby affect its biological function and prevent phage DNA entry [29]. The first description of a *sie* system of *Lactococcus* was published in 2002 and referred to the P335-type temperate lactococcal bacteriophage Tuc2009 [30]. After integration of the bacteriophage Tuc2009 genome into the lactococcal chromosome, the prophage protein Sie2009 is produced and blocks superinfecting phage DNA entry into the cell. The blocking mechanism has not been fully elucidated; nevertheless, it has been proposed that Sie2009 interacts with factor(s) responsible for initiating the phage DNA release from the capsid. Alternatively, the Sie2009 protein might interact with cell membrane proteins that are essential for DNA translocation. The effect of Sie2009 seems to be analogous to the effect of the lysogenic phage repressor (CI) preventing re-infection. In contrast, the presence of the *sie2009* gene determines resistance to various phages, also to phages from other species [28,30]. Similarly to lactococci, in lactobacilli prophages are also a common phenomenon [31]. Comparative genomics of lactobacilli revealed the presence of genes coding for putative proteins with a close sequence match to a surface-exposed lipoprotein encoded by bacteriophage TP-J34 of *Streptococcus thermophilus*, another bacterial species used in industrial milk fermentation processes. The TP-J34 prophage carries a Sie-like system consisting of the *ltp* (lipoprotein of temperate phage) gene, encoding a surface-exposed lipoprotein of biologically proven phage-resistance functions. In view of the fact that the *sie* genes of lactic acid bacteria are located on lysogeny modules of prophages and confer infection exclusion, they have been termed phage-derived phage resistance systems [32].

### **6.3. Restriction modification systems**

28 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

limited to a narrow range of dairy products [25-26].

**6.2. Blocking of phage DNA injection** 

see section 12.2.).

absence in the cell envelopes [24]. Lack of a functional receptor might be due to spontaneous mutations in the genetic material, leading in turn to bacteriophage insensitive mutants (BIM). A good illustration of the BIM phenomenon is a lactococcal mutant in the chromosomally-encoded *pip* gene. The resultant strain is unable to interact with phages of the c2 group, revealing high level of c2-specific resistance [24] (for further details on BIMs

Mechanisms preventing phage adsorption are not only mediated by the bacterial chromosome, but also by acquired plasmids. The best documented plasmid-encoded mechanisms of inhibition of phage adsorption rely on either direct synthesis of cell surface antigens or the production of extracellular carbohydrates. Of the two modes of action, the former reveals phage specificity, whereas the latter seems to restrict access to the bacterial cell for various harmful factors, including bacteriophages [25]. Studies carried by Tuncer and Akcelic demonstrated that a 28.5-kb plasmid, isolated from *L. lactis* subsp. *lactis* MPL56, causes complete inhibition of four lactococcal phages due to the production of a 55.4-kDa protein [25]. The protein exhibits similarity to lectins, a group of proteins that adsorb to specific monosaccharide components of polysaccharides in the cell wall, hence, impairing specific recognition of the phage receptor sites by these four phages. Thus, this plasmidencoded 55.4-kDa protein shields specifically the galactose-containing receptor rather than interacts with the phage, in other words, the bacterial lectin and the phage RBP compete for the receptor [25]. Another example of physical masking of the receptor is the plasmidmediated production of extracellular carbohydrates, called exopolysaccharides (EPS) [26]. Such EPS envelope coats the cell surface giving bacteria extra protection, not only against bacteriophages, but also against desiccation. There is some evidence that EPSs contain sugar residues that are similar or even identical to initial phage receptors. Therefore, phage insensitivity of LAB strains that carry EPS-encoding plasmids, for instance, pCI658, might be due to phage immobilization by binding to EPS [26]. On the other hand, polysaccharides have an impact on the properties of dairy products, like: texture, viscosity and smoothness of mouthfeel. Thereby, application of EPS-producing phage-resistant strains might be

After phage binding to the receptor, phage DNA is introduced into the bacterial cell. In the cytoplasm, phage genetic information is amplified and consequently progeny particles are produced. However, studies of Watanabe on the interaction between phage PL-1 and a *Lactobacillus casei* strain showed no bacterial lysis, despite phage adsorption to cell envelopes [27]. An electron microscopy image indicated that the phage DNA remains intact in the capsid. In contrast to this, a significant increase in the number of empty capsids was observed on the surface of the sensitive strain. In the light of this evidence, it is obvious that phage DNA injection might be interrupted, although the adsorption of phages to the cell surface occurred. Intensive attempts to elucidate the injection blocking phenomenon have allowed identifying different Sie (superinfection exclusion) or Sie-like systems. On the other Following successful injection of DNA, phage infection might be completed or hindered by the presence of restriction modification systems (RM). RM systems comprise two activities represented by the following enzymes: endonuclease (restriction) and methyltransferase (modification) [33]. Simultaneously, both activities are specific to the same target sequences. The endonucleolytic activity is responsible for degradation of invading foreign DNA, including phage DNA, which lack a unique methylation pattern, while the methyltransferase activity protects the host DNA against degradation by introducing a methyl group into a specific nucleotide of the target site [34]. In detail, phage DNA usually reveal different methylation patterns than those recognized by innate RM systems. Unmethylated target sequences are significantly susceptible to endonucleolytic attack,

resulting in DNA degradation [35]. Such mode of action guarantees that the presence of RM systems limits phage proliferation in the cytoplasm, causing no harm to the cell. RM systems are classified into four groups, based on their molecular structure, co-factor requirements, sequence recognition and cleavage position [34-36].

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

located at N and C termini have been proposed to form a split domain, which makes contact

Type I systems have been further classified into four families based on genetic and biochemical criteria, such as: gene order, identity at amino acid level, complementation assay and enzymatic properties. RM systems belonging to type IA, IB, and ID are only chromosomally-encoded, while most complete type IC systems are either chromosomal or carried on large conjugative plasmids [36]. Additionally, numerous small plasmids carry the *hsdS* gene alone [34,40]. While all subunits belong to the same subtype, a plasmid-encoded HsdS protein is able to form a multifunctional enzyme with chromosomally-encoded HsdM and HsdR subunits [41]. Thus, acquisition of a new *hsdS,* revealing new sequence specificity,

Among LAB, type IC systems seem to be most widespread. Type IC RM loci of both *L. lactis*  IL1403 and *L. cremoris* MG1363 consist of three genes: *hsdR*, *hsdM*, *hsdS,* and two promoters, one for transcription of *hsdR* and the other for transcription of both *hsdM* and *hsdS* [17,42]. Nevertheless, there is no clear evidence for transcription regulation of type I RM enzymes [42]. Under these circumstances, an unmodified chromosome is exposed to endonucleolytic digestion after acquisition of either a new system or just the subunit specificity genes. It was observed that a delay in the appearance of restriction activity, which ensures the survival of recipient cells in the absence of complete modification of chromosomal target sites, depends on host function [36,43]. Chromosomally-encoded energy-dependent proteases ClpP and ClpX, co-operating in a complex, are implicated in the regulation of restriction activity [36]. The ClpXP complex is responsible for restriction alleviation through proteolytic degradation of HsdR subunits. Based on results of Janscak and colleagues concerning the EcoR124I endonuclease, an alternative mechanism of delay in restriction alleviation has been proposed. As each of the two HsdR subunits interacts differently with HsdM, it has been postulated that the control of restriction activity is implemented at the level of subunit assembly [38]. Formation of a weak R2M2S1 restriction complex will be suspended, unless accumulation of HsdR molecules occurs. Excess of HsdR over HsdM is observed in the late stage of establishing of the RM system in a recipient cell; hence, the unmodified

In contrast to type I, type II RM systems are structurally the simplest of all restriction modification systems. They are generally encoded by two genes, but the key defining feature of this RM type is the independent activity of restriction and modification enzymes [33]. Methyltransferase is active as an asymmetric monomer, requires only AdoMet, and recognizes the same target sequences as the cognate endonuclease. In contrast, restriction endonuclease is a homodimer and requires divalent Mg2+ cations for proper activity. Endonucleases generally recognize a palindromic 4-8 bp DNA sequence and cleave within or in a fixed distance of the recognition site. In contrast to type I, ATP has no effect on the

chromosome is protected against premature restriction activity [38].

cleavage activity of type II endonucleases [44].

with a second HsdM subunit [35,37].

leads to the increase of phage resistance.

*6.3.2. Type II RM* 

to Lower Phage Contamination in Dairy Fermentation 31
