*6.3.1. Type I RM*

Type I is the most complex RM system in terms of genetic organization and biochemical activity. It is composed of three different *hsd* (host specificity determinant) genes coding for the following subunits: HsdR - responsible for restriction, HsdM - involved in modification and HsdS - responsible for specific sequence recognition. None of them reveals any activity as a single protein [36]. In order for the modification activity to occur, a combination of one HsdS and two HsdM subunits is required. The M2S1 multifunctional enzyme acts as protective methyltransferase, which modifies DNA through the transfer of the methyl group from S-Adenosyl-methionine (AdoMet) to the specific adenines in the recognition site [36,38]. For restriction activity, all subunits are absolutely required in a stoichiometric ratio of R2M2S1. This holoenzyme exhibits both endonucleolytic and helicase activities, and is active only in presence of Mg2+, AdoMet and ATP [36].

Besides the complex structure of this multifunctional enzyme, also structure of the recognized sequences and cleavage position are the distinguishing features of type I RM systems. Type I RM enzymes specifically recognize asymmetric and bipartite sequences. These non-palindromic DNA sequences consist of two specific components, one of 3-4 bp and the other of 4-5 bp, separated by a 6-8 bp non-specific sequence [34,36-37]. The innate methylation state of the target sequence determines the activity of the multifunctional R2M2S1 enzyme. When the target sequence is methylated or semi-methylated (e.g. just after replication), the enzyme will exhibit activity of a methyltransferase, which completes DNA modification. In contrast, if the holoenzyme binds to an unmethylated recognition site, DNA translocation past the DNA-enzyme complex occurs in an ATP-dependent manner [35,38]. In spite of DNA translocation, the enzyme remains bound to the target site. DNA is cleaved at a position, where either collision with another translocating complex has appeared or translocation is halted due to the topology of the DNA substrate. Consequently, type I restriction enzymes cleave DNA randomly at a nonspecific site, far from the recognition sequence [38].

Interaction between subunits, leading to formation of multifunctional enzymes as well as interaction of resultant enzyme molecules with DNA, are determined by the structure of the HsdS subunits. HsdS subunits consist of regions, which amino acid sequences are conserved within an enzyme family, and two independent target recognition domains (TRD) that share low level of amino acid identity [34,39]. TRDs are involved in target sequence recognition, each TRD recognizes one-half of the split target site and is responsible for DNA binding. Since TRDs are highly variable, they recognize multiple target sequences, and thus, provide a variety of phage resistance types [34,36,39]. The central domain, located between two TRDs, is responsible for interaction with one HsdM subunit. Other conserved regions located at N and C termini have been proposed to form a split domain, which makes contact with a second HsdM subunit [35,37].

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, leads to the increase of phage resistance.

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 chromosome is protected against premature restriction activity [38].

### *6.3.2. Type II RM*

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

sequence recognition and cleavage position [34-36].

active only in presence of Mg2+, AdoMet and ATP [36].

*6.3.1. Type I RM* 

sequence [38].

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,

Type I is the most complex RM system in terms of genetic organization and biochemical activity. It is composed of three different *hsd* (host specificity determinant) genes coding for the following subunits: HsdR - responsible for restriction, HsdM - involved in modification and HsdS - responsible for specific sequence recognition. None of them reveals any activity as a single protein [36]. In order for the modification activity to occur, a combination of one HsdS and two HsdM subunits is required. The M2S1 multifunctional enzyme acts as protective methyltransferase, which modifies DNA through the transfer of the methyl group from S-Adenosyl-methionine (AdoMet) to the specific adenines in the recognition site [36,38]. For restriction activity, all subunits are absolutely required in a stoichiometric ratio of R2M2S1. This holoenzyme exhibits both endonucleolytic and helicase activities, and is

Besides the complex structure of this multifunctional enzyme, also structure of the recognized sequences and cleavage position are the distinguishing features of type I RM systems. Type I RM enzymes specifically recognize asymmetric and bipartite sequences. These non-palindromic DNA sequences consist of two specific components, one of 3-4 bp and the other of 4-5 bp, separated by a 6-8 bp non-specific sequence [34,36-37]. The innate methylation state of the target sequence determines the activity of the multifunctional R2M2S1 enzyme. When the target sequence is methylated or semi-methylated (e.g. just after replication), the enzyme will exhibit activity of a methyltransferase, which completes DNA modification. In contrast, if the holoenzyme binds to an unmethylated recognition site, DNA translocation past the DNA-enzyme complex occurs in an ATP-dependent manner [35,38]. In spite of DNA translocation, the enzyme remains bound to the target site. DNA is cleaved at a position, where either collision with another translocating complex has appeared or translocation is halted due to the topology of the DNA substrate. Consequently, type I restriction enzymes cleave DNA randomly at a nonspecific site, far from the recognition

Interaction between subunits, leading to formation of multifunctional enzymes as well as interaction of resultant enzyme molecules with DNA, are determined by the structure of the HsdS subunits. HsdS subunits consist of regions, which amino acid sequences are conserved within an enzyme family, and two independent target recognition domains (TRD) that share low level of amino acid identity [34,39]. TRDs are involved in target sequence recognition, each TRD recognizes one-half of the split target site and is responsible for DNA binding. Since TRDs are highly variable, they recognize multiple target sequences, and thus, provide a variety of phage resistance types [34,36,39]. The central domain, located between two TRDs, is responsible for interaction with one HsdM subunit. Other conserved regions 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 cleavage activity of type II endonucleases [44].

As this RM type is more heterogeneous in respect to endonucleolytic activity than originally thought based on their structural simplicity, the described mode of action refers mainly to typical (orthodox) type II endonucleases [45].

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

Unlike types I and II, type III systems are less spread among lactic acid bacteria. The *Lla*FI system identified on the lactococcal pND801 plasmid is the first type III RM system described not just in LAB, but generally in Gram-positive bacteria [51]. Based on computational analyses of genome sequences, type III systems were observed to occur also in lactobacilli (for instance *Lactobacillus johnsonii* and *Lb. rhamnosus*) [52]. On the one hand, type III resemble type II systems in their structural and genetic organization. Type III, like type II systems, consists of two genes, one encoding a methyltransferase (Mod) and the other - an endonuclease (Res). Mod is responsible for binding and methylating the recognition sequences, regardless of the presence of Res. On the other hand, type III systems are similar to type I, in respect to endonuclease activity, as the Res subunit is only active in a complex with Mod. Another basic similarity to type I systems is the fact that they both comprise the helicase domain and require both AdoMet and ATP for full restriction activity. The distinctive features characterizing type III systems concern recognition sequences and cleavage sites. The Mod subunit recognizes asymmetric, opposite-oriented sequences and methylation takes place only on one strand of the DNA [53]. The Res endonuclease cuts both strands of the DNA at the distance of 24-27 nucleotides downstream of the unmethylated

Lactococci have been found to possess three types of RM systems: type I, II and III. Based on genomic sequence data, it is evident that RM genes are both chromosomally- and plasmidencoded. However, a variety of RM determinants is generally associated with plasmids [17]. In contrast, very few phage defense mechanisms have been described for *S. thermophilus*. In 2001, Solow and Somkuti reported on the discovery of a complete type I RM system encoded on a streptococcal plasmid pER35 [54]. Further progress in genome sequencing led to finding complete type I and III RM systems in chromosomes of *S. thermophilus* strains. Genome sequence analyses revealed that lactobacilli, like lactococci and streptococci,

To date, no type IV RM systems has been distinguished in lactic acid bacteria. It is highly likely that in the future members of this class will be discovered in LAB. For that reason as well as from the evolutionary point of view, the type IV RM system is worth mentioning. A fusion of genes coding for Mod and Res subunits of type III systems was a key step for evolution of type IV RM [56]. The resulting endonuclease (revealing also methyltransferase activity) has an asymmetrical recognition sequence and cleavage occurs at a fixed distance from the recognition site, like for the type IIS enzymes. On the other hand, this endonuclease requires AdoMet, which distinguishes it from type II endonuclease activity. Therefore, taking into account the enzymatic features of model type IV *Eco*57I and *Bse*MII endonucleases, it has been hypothesized that type IV endonucleases are an intermediate

possess in their chromosomes three types (I-III) of RM systems [55].

*6.3.3. Type III RM* 

specific sites [53].

*6.3.4. Type IV RM* 

between type III and type IIS enzymes.

to Lower Phage Contamination in Dairy Fermentation 33

Apart from the orthodox type (called IIP ), type II restriction enzymes have been categorized into the following subclasses: IIA, IIB, IIC, IIE, IIF, IIG, IIH, IIM, IIS and IIT. Endonucleases of these subclasses differ in structure of the recognized sequence (asymmetric or symmetric), cleavage positions and cofactor requirements. Type IIA endonucleases behave similarly to the orthodox class, but recognize asymmetric sequences [45]. The unique feature of subclass IIB refers to the cleavage position. These endonucleases cut DNA from both sides, which results in complete extraction of the target sequence from the DNA molecule [46]. Subclasses IIC and IIE have both modification and restriction domains present in one polypeptide. Additionally, class IIE endonucleases interact with two copies of their recognition site, one copy being the target for cleavage, the other serving as an allosteric effector [47]. Similarly to subclass IIE, class IIF restriction enzymes interact with two copies of their recognition sequences, but cleavage occurs at both sequences. Type IIG restriction enzymes seem to combine properties of both IIB and IIC subclasses. The methyltransferase activity of class IIG, like IIB, is stimulated by AdoMet. The main similarity between IIG and IIC is that they both have restriction and modification activities located on one polypeptide chain [45,47]. Subclass IIH, represented by the AhdI system, appears to be a novel RM system due to its genetic organization resembling that of type I. As in type II systems, the AhdI endonuclease is encoded by a single gene; on the other hand, similarly to type I, its cognate methyltransferase forms a complex consisting of two modification and two specificity subunits [44,48]. Subclass IIM is at the opposite extreme from other type II subclasses as it recognizes and cleavages methylated target sequences. The key distinguishing feature of type IIS is the cleavage position outside of the recognition sequence at a defined distance [49]. Subclass IIT is an example of a variation in the typical genetic organization of type II RM systems, as the endonuclease is composed of two different subunits. Moreover, some IIT endonucleases function not only as heterodimers, but also as heterotetramers [44-45].

As enzymes belonging to type II systems are the most abundant and mainly encoded on plasmids, they can be acquired by the bacterial cell through plasmid transfer events. Therefore, a question arises as how to protect the host cell against an incoming endonuclease. In many cases, each gene of the type II RM system has its own promoter. Thus, a delay in appearance of the endonuclease activity is regulated at the transcriptional level. The lactococcal LlaDII RM system is a good example which illustrates this type of regulation [50]. At the initial stage of establishing in the host cell, the LlaDII methyltransferase is overexpressed, whereas the restriction enzyme is produced in small amounts due to the weak constitutive expression of its gene. On the other hand, a permanently high concentration of methylases is an unfavorable circumstance due to possible methylation and therefore protection of the invading phage DNA. The LlaDII methyltransferase contains HTH motifs, which were shown to be engaged in direct interaction with its promoter sequence, causing silencing of its own gene expression [50].

### *6.3.3. Type III RM*

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

typical (orthodox) type II endonucleases [45].

but also as heterotetramers [44-45].

As this RM type is more heterogeneous in respect to endonucleolytic activity than originally thought based on their structural simplicity, the described mode of action refers mainly to

Apart from the orthodox type (called IIP ), type II restriction enzymes have been categorized into the following subclasses: IIA, IIB, IIC, IIE, IIF, IIG, IIH, IIM, IIS and IIT. Endonucleases of these subclasses differ in structure of the recognized sequence (asymmetric or symmetric), cleavage positions and cofactor requirements. Type IIA endonucleases behave similarly to the orthodox class, but recognize asymmetric sequences [45]. The unique feature of subclass IIB refers to the cleavage position. These endonucleases cut DNA from both sides, which results in complete extraction of the target sequence from the DNA molecule [46]. Subclasses IIC and IIE have both modification and restriction domains present in one polypeptide. Additionally, class IIE endonucleases interact with two copies of their recognition site, one copy being the target for cleavage, the other serving as an allosteric effector [47]. Similarly to subclass IIE, class IIF restriction enzymes interact with two copies of their recognition sequences, but cleavage occurs at both sequences. Type IIG restriction enzymes seem to combine properties of both IIB and IIC subclasses. The methyltransferase activity of class IIG, like IIB, is stimulated by AdoMet. The main similarity between IIG and IIC is that they both have restriction and modification activities located on one polypeptide chain [45,47]. Subclass IIH, represented by the AhdI system, appears to be a novel RM system due to its genetic organization resembling that of type I. As in type II systems, the AhdI endonuclease is encoded by a single gene; on the other hand, similarly to type I, its cognate methyltransferase forms a complex consisting of two modification and two specificity subunits [44,48]. Subclass IIM is at the opposite extreme from other type II subclasses as it recognizes and cleavages methylated target sequences. The key distinguishing feature of type IIS is the cleavage position outside of the recognition sequence at a defined distance [49]. Subclass IIT is an example of a variation in the typical genetic organization of type II RM systems, as the endonuclease is composed of two different subunits. Moreover, some IIT endonucleases function not only as heterodimers,

As enzymes belonging to type II systems are the most abundant and mainly encoded on plasmids, they can be acquired by the bacterial cell through plasmid transfer events. Therefore, a question arises as how to protect the host cell against an incoming endonuclease. In many cases, each gene of the type II RM system has its own promoter. Thus, a delay in appearance of the endonuclease activity is regulated at the transcriptional level. The lactococcal LlaDII RM system is a good example which illustrates this type of regulation [50]. At the initial stage of establishing in the host cell, the LlaDII methyltransferase is overexpressed, whereas the restriction enzyme is produced in small amounts due to the weak constitutive expression of its gene. On the other hand, a permanently high concentration of methylases is an unfavorable circumstance due to possible methylation and therefore protection of the invading phage DNA. The LlaDII methyltransferase contains HTH motifs, which were shown to be engaged in direct interaction with its promoter sequence, causing silencing of its own gene expression [50].

Unlike types I and II, type III systems are less spread among lactic acid bacteria. The *Lla*FI system identified on the lactococcal pND801 plasmid is the first type III RM system described not just in LAB, but generally in Gram-positive bacteria [51]. Based on computational analyses of genome sequences, type III systems were observed to occur also in lactobacilli (for instance *Lactobacillus johnsonii* and *Lb. rhamnosus*) [52]. On the one hand, type III resemble type II systems in their structural and genetic organization. Type III, like type II systems, consists of two genes, one encoding a methyltransferase (Mod) and the other - an endonuclease (Res). Mod is responsible for binding and methylating the recognition sequences, regardless of the presence of Res. On the other hand, type III systems are similar to type I, in respect to endonuclease activity, as the Res subunit is only active in a complex with Mod. Another basic similarity to type I systems is the fact that they both comprise the helicase domain and require both AdoMet and ATP for full restriction activity. The distinctive features characterizing type III systems concern recognition sequences and cleavage sites. The Mod subunit recognizes asymmetric, opposite-oriented sequences and methylation takes place only on one strand of the DNA [53]. The Res endonuclease cuts both strands of the DNA at the distance of 24-27 nucleotides downstream of the unmethylated specific sites [53].

Lactococci have been found to possess three types of RM systems: type I, II and III. Based on genomic sequence data, it is evident that RM genes are both chromosomally- and plasmidencoded. However, a variety of RM determinants is generally associated with plasmids [17]. In contrast, very few phage defense mechanisms have been described for *S. thermophilus*. In 2001, Solow and Somkuti reported on the discovery of a complete type I RM system encoded on a streptococcal plasmid pER35 [54]. Further progress in genome sequencing led to finding complete type I and III RM systems in chromosomes of *S. thermophilus* strains. Genome sequence analyses revealed that lactobacilli, like lactococci and streptococci, possess in their chromosomes three types (I-III) of RM systems [55].

### *6.3.4. Type IV RM*

To date, no type IV RM systems has been distinguished in lactic acid bacteria. It is highly likely that in the future members of this class will be discovered in LAB. For that reason as well as from the evolutionary point of view, the type IV RM system is worth mentioning. A fusion of genes coding for Mod and Res subunits of type III systems was a key step for evolution of type IV RM [56]. The resulting endonuclease (revealing also methyltransferase activity) has an asymmetrical recognition sequence and cleavage occurs at a fixed distance from the recognition site, like for the type IIS enzymes. On the other hand, this endonuclease requires AdoMet, which distinguishes it from type II endonuclease activity. Therefore, taking into account the enzymatic features of model type IV *Eco*57I and *Bse*MII endonucleases, it has been hypothesized that type IV endonucleases are an intermediate between type III and type IIS enzymes.

In summary, it has been well documented that phage restriction-modification systems are widely spread among lactic acid bacteria. Nevertheless, comparative genomics of LAB demonstrated that bacteria representing different niches vary in the presence of restrictionmodification genes. The lack of RM systems is a common feature for LAB isolated from the gut, whereas the presence of RM genes is a typical feature for dairy species. Therefore, it was proposed that genes constituting the restriction-modification systems, together with certain genes of sugar metabolism and the proteolytic system, constitute "a barcode" of genes, which can indicate the ability of the microorganism to occupy either dairy or gut niches [57].

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

particles before completion of the maturation process. The timing of phage lysis is controlled by the phage holin protein; thus, AbiZ might interact cooperatively with the

While the mechanism of cell death in the AbiZ system is self-explanatory, in case of other Abi systems is poorly elucidated. The most likely explanation for this phenomenon is that Abi proteins interfere with processes essential not only for phage, but also for bacterial development; therefore, death of individual bacterial cells is always observed following activation of the Abi systems [17,58-59]. As a consequence, release of progeny particles is limited and the bacterial population survives. Hence, the Abi systems constitute a barrier against bacteriophage proliferation, in which "altruistic suicide" of infected bacterial cells

Another naturally-occurring distinct phage defense system recently described in Prokaryotes is CRISPR/*cas*. Besides RM mechanisms, this system is also directly engaged in protecting bacterial cells against invading genetic elements, such as phages or plasmids [66]. In brief, CRISPR-conferred phage resistance relies on incorporation of short phage-derived sequences within specific loci of the bacterial genome. In effect, the bacterial cell becomes

CRISPR/*cas* systems are composed of two specific determinants: (i) clustered regularly interspaced short palindromic regions (CRISPR array) and (ii) regions encoding CRISPRassociated (Cas) proteins. The CRISPR arrays consist of non-coding sequences composed of unique phage-derived spacers (21-72 bp) separated by short direct repeated sequences (21- 48 bp) of bacterial origin. The length of spacers and repeats within a single array is always the same, while their number may vary from 2-375, depending on the species. On the other hand, Cas proteins constitute a heterologous group of proteins, which contain various functional domains, e.g. typical for nucleases, helicases, nucleic acid binding proteins, etc. [66]. The specific role of individual Cas proteins vary as they were shown to be engaged at various stages of CRISPR-conferred resistance. Interestingly, *cas* genes were detected only in CRISPR-containing genomes, suggesting their tight association. The number of *cas* genes within a CRISPR locus varies from 4 to 20 [67]. Their position can be either upstream or downstream of repeat-spacer units, but always from the same side for a given CRISPR locus type. The CRISPR array and Cas-encoding genes are separated by an A-T rich leader region, suggested to be the promoter region of CRISPR transcription; yet, mechanisms regulating expression still remain to be elucidated [68]. Together these two elements, CRISPR spacerrepeat array and Cas proteins, provide "immunity" to the bacterial cell against invading foreign DNA molecules, including phages (for detailed review see: [67-69]). CRISPR arrays are widely distributed within the Prokaryotic world and are detected in the genomes of 40% of Bacteria and 90% of Archea [70]. Depending on the species, a single genome can carry up

to 18 CRISPR loci, which are suggested to confer resistance to various phages [66].

phage holin or with a holin inhibitor to make it active prematurely [59].

provides protection of the whole uninfected population [17,58].

immune to phages which carry homologous sequences.

**6.5. CRISPR/***cas* **systems** 

to Lower Phage Contamination in Dairy Fermentation 35

### **6.4. Phage abortive infection systems**

When the RM systems fail in protecting the bacterium against invading phage DNA, initiation of the phage propagation cycle occurs. However, proliferation of progeny particles might be dramatically limited due to systems that abort the infection at various points of the phage cycle. Abortive infection mechanisms (Abi) have different targets in the cell. They are able to interrupt phage DNA replication, transcription, protein synthesis, phage particle assembly or induce premature cell lysis [17,58]. The Abi mechanisms have been found in many bacterial species, including *Escherichia coli*, *Bacillus subtilis*, *Streptococcus pyogenes*, *Vibrio cholerae* and *Lactococcus lactis* [58]. The most known Abis have been found in the latter species. To date, 22 lactococcal Abi mechanisms have been identified and designated into various groups distinguished by a subsequent letter of the alphabet [58-60]. Most of Abi systems are plasmid-encoded and only three are located on chromosomal DNA (*abi*H, *abi*N, *abi*V) [60]. For instance, *abi*N is located in a prophage region of the *L. lactis* subsp*. cremoris* MG1364 genome and exhibits significant similarity to a corresponding region of the lactococcal temperate phage rlt [61]. Abi systems present simple genetic organization. The Abi phenotype is most frequently encoded by a single gene; however, more complex structures have been identified in six systems. AbiE, AbiG, AbiL, AbiT and AbiU are encoded by two genes, whereas AbiR is the only system identified until now that is encoded by three separate genes [58, 62-63]. Proteins encoded by *abi* genes are cytoplasm-located, where they reveal their activity. In contrast, the AbiP system is represented by a membraneanchored protein [64].

Abi systems reveal a variety of modes of action. However, in many cases, mechanisms of action of the individual systems were not fully elucidated. Some Abis, like AbiA, AbiD1, AbiF, AbiK, AbiP and AbiT, have been found to interfere with DNA replication, whereas AbiB, AbiG and AbiU arrest mRNA synthesis or have a negative impact on stabilization of transcripts. Haaber and colleagues presented that the AbiV system strongly affects translation of both early and late phage proteins, shortly after infection. Based on this observation, it was concluded that the AbiV system arrests the bacterial translation apparatus [60]. AbiE, AbiI, AbiQ and AbiZ systems affect maturation of phage particles [59,65]. The AbiZ system, identified in 2007 by Durmaz and Klaenhammer, induces premature lysis of phage-infected cells, resulting in the release of the developing phage particles before completion of the maturation process. The timing of phage lysis is controlled by the phage holin protein; thus, AbiZ might interact cooperatively with the phage holin or with a holin inhibitor to make it active prematurely [59].

While the mechanism of cell death in the AbiZ system is self-explanatory, in case of other Abi systems is poorly elucidated. The most likely explanation for this phenomenon is that Abi proteins interfere with processes essential not only for phage, but also for bacterial development; therefore, death of individual bacterial cells is always observed following activation of the Abi systems [17,58-59]. As a consequence, release of progeny particles is limited and the bacterial population survives. Hence, the Abi systems constitute a barrier against bacteriophage proliferation, in which "altruistic suicide" of infected bacterial cells provides protection of the whole uninfected population [17,58].

### **6.5. CRISPR/***cas* **systems**

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

**6.4. Phage abortive infection systems** 

niches [57].

anchored protein [64].

In summary, it has been well documented that phage restriction-modification systems are widely spread among lactic acid bacteria. Nevertheless, comparative genomics of LAB demonstrated that bacteria representing different niches vary in the presence of restrictionmodification genes. The lack of RM systems is a common feature for LAB isolated from the gut, whereas the presence of RM genes is a typical feature for dairy species. Therefore, it was proposed that genes constituting the restriction-modification systems, together with certain genes of sugar metabolism and the proteolytic system, constitute "a barcode" of genes, which can indicate the ability of the microorganism to occupy either dairy or gut

When the RM systems fail in protecting the bacterium against invading phage DNA, initiation of the phage propagation cycle occurs. However, proliferation of progeny particles might be dramatically limited due to systems that abort the infection at various points of the phage cycle. Abortive infection mechanisms (Abi) have different targets in the cell. They are able to interrupt phage DNA replication, transcription, protein synthesis, phage particle assembly or induce premature cell lysis [17,58]. The Abi mechanisms have been found in many bacterial species, including *Escherichia coli*, *Bacillus subtilis*, *Streptococcus pyogenes*, *Vibrio cholerae* and *Lactococcus lactis* [58]. The most known Abis have been found in the latter species. To date, 22 lactococcal Abi mechanisms have been identified and designated into various groups distinguished by a subsequent letter of the alphabet [58-60]. Most of Abi systems are plasmid-encoded and only three are located on chromosomal DNA (*abi*H, *abi*N, *abi*V) [60]. For instance, *abi*N is located in a prophage region of the *L. lactis* subsp*. cremoris* MG1364 genome and exhibits significant similarity to a corresponding region of the lactococcal temperate phage rlt [61]. Abi systems present simple genetic organization. The Abi phenotype is most frequently encoded by a single gene; however, more complex structures have been identified in six systems. AbiE, AbiG, AbiL, AbiT and AbiU are encoded by two genes, whereas AbiR is the only system identified until now that is encoded by three separate genes [58, 62-63]. Proteins encoded by *abi* genes are cytoplasm-located, where they reveal their activity. In contrast, the AbiP system is represented by a membrane-

Abi systems reveal a variety of modes of action. However, in many cases, mechanisms of action of the individual systems were not fully elucidated. Some Abis, like AbiA, AbiD1, AbiF, AbiK, AbiP and AbiT, have been found to interfere with DNA replication, whereas AbiB, AbiG and AbiU arrest mRNA synthesis or have a negative impact on stabilization of transcripts. Haaber and colleagues presented that the AbiV system strongly affects translation of both early and late phage proteins, shortly after infection. Based on this observation, it was concluded that the AbiV system arrests the bacterial translation apparatus [60]. AbiE, AbiI, AbiQ and AbiZ systems affect maturation of phage particles [59,65]. The AbiZ system, identified in 2007 by Durmaz and Klaenhammer, induces premature lysis of phage-infected cells, resulting in the release of the developing phage Another naturally-occurring distinct phage defense system recently described in Prokaryotes is CRISPR/*cas*. Besides RM mechanisms, this system is also directly engaged in protecting bacterial cells against invading genetic elements, such as phages or plasmids [66]. In brief, CRISPR-conferred phage resistance relies on incorporation of short phage-derived sequences within specific loci of the bacterial genome. In effect, the bacterial cell becomes immune to phages which carry homologous sequences.

CRISPR/*cas* systems are composed of two specific determinants: (i) clustered regularly interspaced short palindromic regions (CRISPR array) and (ii) regions encoding CRISPRassociated (Cas) proteins. The CRISPR arrays consist of non-coding sequences composed of unique phage-derived spacers (21-72 bp) separated by short direct repeated sequences (21- 48 bp) of bacterial origin. The length of spacers and repeats within a single array is always the same, while their number may vary from 2-375, depending on the species. On the other hand, Cas proteins constitute a heterologous group of proteins, which contain various functional domains, e.g. typical for nucleases, helicases, nucleic acid binding proteins, etc. [66]. The specific role of individual Cas proteins vary as they were shown to be engaged at various stages of CRISPR-conferred resistance. Interestingly, *cas* genes were detected only in CRISPR-containing genomes, suggesting their tight association. The number of *cas* genes within a CRISPR locus varies from 4 to 20 [67]. Their position can be either upstream or downstream of repeat-spacer units, but always from the same side for a given CRISPR locus type. The CRISPR array and Cas-encoding genes are separated by an A-T rich leader region, suggested to be the promoter region of CRISPR transcription; yet, mechanisms regulating expression still remain to be elucidated [68]. Together these two elements, CRISPR spacerrepeat array and Cas proteins, provide "immunity" to the bacterial cell against invading foreign DNA molecules, including phages (for detailed review see: [67-69]). CRISPR arrays are widely distributed within the Prokaryotic world and are detected in the genomes of 40% of Bacteria and 90% of Archea [70]. Depending on the species, a single genome can carry up to 18 CRISPR loci, which are suggested to confer resistance to various phages [66].

The mechanism of CRISPR/*cas* conferred protection of bacterial cells against phage infection is rather complex and can be divided into three main stages: (i) adaptation, (ii) CRISPR expression and (iii) CRISPR-mediated interference. The first stage relies on incorporation into the bacterial genome within the CRISPR locus of short phage-derived fragments (protospacers). Despite the fact that the exact mechanism of spacer acquisition is not known, it is not accidental. Recognition of specific phage sequences for integration is suggested to be linked with sequences termed PAMs (proto-spacer adjacent motifs), located up- or downstream of the proto-spacer. Integration of new spacers occurs from the end of the leader region, between the palindromic repeats and involves certain Cas proteins. Stage 2 is CRISPR expression, which involves transcription of the whole CRISPR spacer-repeat array (pre-mRNA). The presence of palindromic repeat sequences within the transcript, leads to formation of secondary hair-pin like structures. These are subsequently processed into short CRISPR RNAs (crRNAs) by endonucleolytic digestion at a cleavage site located downstream from the last nucleotide forming the hairpin. Finally, the last stage of CRISPR/*cas* activity is based on interaction of mature crRNAs with invading foreign DNA elements (phages), which leads to silencing/degradation of the latter by a certain group of Cas proteins. By this activity, CRISPR/*cas*-carrying hosts are protected from invasion by phages carrying sequences homologous to the integrated spacers. Application of the CRISPR/*cas* system for developing novel phage resistant dairy starter strains may be an attractive alternative, which will be discussed in further parts of this chapter (see section: 12.4.).

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

system interferes with the phage life cycle, inhibiting translation of essential phage genes or

An example are systems developed in *Streptococcus thermophilus,* which were shown to provide protection against Sfi21-type phages, including 3 [73-74]. These systems are based on expression of antisense RNAs against genes from the replication module of the Sfi21-type phage 3 genome, e.g. putative primase (*pri3.1*) or helicase (*hel3.1*) genes. Strong conservation of the whole replication module among the already sequenced Sfi21-type phages makes it a good target for inhibiting phage development [73]. Moreover, hybridization studies revealed that the Sfi21-type replication module is commonly encountered in majority of industrially isolated phages. This reinforced the choice to use it

To test the efficiency of the Sfi21-type module antisense RNA system, constructs expressing antisense RNA cassettes of different length were introduced into *S. thermophilus* strains, which were then challenged with phage infection. The most effective were constructs expressing antisense RNA covering the whole region of target (primase or helicase gene). Also shorter RNAs provided sufficient phage resistance, which was speculated to be due to the presence of specific structural or potential regulatory domains within these fragments. Furthermore, in case of constructs harboring antisense RNAs of similar length, more efficient were usually those comprising the RBS (ribosome-binding sequence) sites. Such effect was believed to be due to the fact that the antisense RBS sequences prevented gene translation by impeding efficient loading of ribosomes onto phage mRNAs [72]. Overall, expression of phage antisense RNAs in *S. thermophilus* was shown to interfere/delay the intracellular phage DNA replication, decrease phage plaque formation (EOP, efficiency of plating), lower the abundance of phage sense mRNA transcripts and reduce phage progeny particles released from infected cells [73,75].

Similar systems were also developed in *Lactococcus lactis* by expressing anti-sense RNAs directed against various phage genes (e.g. P335-type *gp18C* and *gp24C, gp15C* alone, or putative replication genes, 936-type phage F4-1 major coat protein (*mcp*) gene) [76-79]. In these cases, similarly as for *S. thermophilus* systems, the most efficient antisense RNAs in

Current data allow to conclude that the most effective antiRNA-based phage defense systems, apart from some exceptions, are those which target: (i) genes vital for phage development (e.g. involved in synthesis of phage DNA), (ii) preferably early-expressed phage genes, (iii) genes expressed at low levels, (iv) genes which respective transcripts are unstable [73,79]. Sequencing of novel phage genomes and development of comparative genomics allows identification of other conserved phage genome regions that could serve as

Defense systems that employ elements derived from lytic phage genomes are termed phageencoded resistance (PER). One type of engineered PER systems is based on the origin (*ori*) of

inhibiting phage development were those comprising the RBS site.

degradation of their mRNAs [72].

as a phage defense element [73,75].

potential targets of antiRNAs.

*6.6.2. Origin-derived phage-encoded resistance* 

to Lower Phage Contamination in Dairy Fermentation 37

### **6.6. Engineered defense systems**

Besides the naturally-occurring defense mechanisms against recurrent phage infections (discussed above), new methods involving molecular techniques are designed to combat phages. The constantly growing knowledge on phage development and their genome sequences allows currently to develop engineered defense systems, which are otherwise not encountered in nature (for review see also: [71]). The idea of such systems relies on engineering bacterial strains in a way which impairs genes vital for phage development, e.g. phage replication proteins or other replication factors. Moreover, identification of homologues of these crucial genes within multiple phage genomes allows creating broadrange phage defense systems. As presented below, numerous studies deliver clear evidence that such engineered systems provide efficient protection against phage infections. The following parts of this chapter will delineate each of these systems in more details. Studies on developing engineered systems for lactic acid bacteria were performed in most part in *Streptococcus thermophilus* and *Lactococcus lactis* as strains from both species find wide applications in dairy fermentation processes.

### *6.6.1. Antisense RNA-based phage defense systems*

Bacterial-engineered expression of antisense RNA directed against phage transcripts has been described as one of the most efficient phage defense systems. The mode of action of such RNAs is hybridization to phage sense strand RNAs upon infection. By these means the system interferes with the phage life cycle, inhibiting translation of essential phage genes or degradation of their mRNAs [72].

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

The mechanism of CRISPR/*cas* conferred protection of bacterial cells against phage infection is rather complex and can be divided into three main stages: (i) adaptation, (ii) CRISPR expression and (iii) CRISPR-mediated interference. The first stage relies on incorporation into the bacterial genome within the CRISPR locus of short phage-derived fragments (protospacers). Despite the fact that the exact mechanism of spacer acquisition is not known, it is not accidental. Recognition of specific phage sequences for integration is suggested to be linked with sequences termed PAMs (proto-spacer adjacent motifs), located up- or downstream of the proto-spacer. Integration of new spacers occurs from the end of the leader region, between the palindromic repeats and involves certain Cas proteins. Stage 2 is CRISPR expression, which involves transcription of the whole CRISPR spacer-repeat array (pre-mRNA). The presence of palindromic repeat sequences within the transcript, leads to formation of secondary hair-pin like structures. These are subsequently processed into short CRISPR RNAs (crRNAs) by endonucleolytic digestion at a cleavage site located downstream from the last nucleotide forming the hairpin. Finally, the last stage of CRISPR/*cas* activity is based on interaction of mature crRNAs with invading foreign DNA elements (phages), which leads to silencing/degradation of the latter by a certain group of Cas proteins. By this activity, CRISPR/*cas*-carrying hosts are protected from invasion by phages carrying sequences homologous to the integrated spacers. Application of the CRISPR/*cas* system for developing novel phage resistant dairy starter strains may be an attractive alternative,

which will be discussed in further parts of this chapter (see section: 12.4.).

Besides the naturally-occurring defense mechanisms against recurrent phage infections (discussed above), new methods involving molecular techniques are designed to combat phages. The constantly growing knowledge on phage development and their genome sequences allows currently to develop engineered defense systems, which are otherwise not encountered in nature (for review see also: [71]). The idea of such systems relies on engineering bacterial strains in a way which impairs genes vital for phage development, e.g. phage replication proteins or other replication factors. Moreover, identification of homologues of these crucial genes within multiple phage genomes allows creating broadrange phage defense systems. As presented below, numerous studies deliver clear evidence that such engineered systems provide efficient protection against phage infections. The following parts of this chapter will delineate each of these systems in more details. Studies on developing engineered systems for lactic acid bacteria were performed in most part in *Streptococcus thermophilus* and *Lactococcus lactis* as strains from both species find wide

Bacterial-engineered expression of antisense RNA directed against phage transcripts has been described as one of the most efficient phage defense systems. The mode of action of such RNAs is hybridization to phage sense strand RNAs upon infection. By these means the

**6.6. Engineered defense systems** 

applications in dairy fermentation processes.

*6.6.1. Antisense RNA-based phage defense systems* 

An example are systems developed in *Streptococcus thermophilus,* which were shown to provide protection against Sfi21-type phages, including 3 [73-74]. These systems are based on expression of antisense RNAs against genes from the replication module of the Sfi21-type phage 3 genome, e.g. putative primase (*pri3.1*) or helicase (*hel3.1*) genes. Strong conservation of the whole replication module among the already sequenced Sfi21-type phages makes it a good target for inhibiting phage development [73]. Moreover, hybridization studies revealed that the Sfi21-type replication module is commonly encountered in majority of industrially isolated phages. This reinforced the choice to use it as a phage defense element [73,75].

To test the efficiency of the Sfi21-type module antisense RNA system, constructs expressing antisense RNA cassettes of different length were introduced into *S. thermophilus* strains, which were then challenged with phage infection. The most effective were constructs expressing antisense RNA covering the whole region of target (primase or helicase gene). Also shorter RNAs provided sufficient phage resistance, which was speculated to be due to the presence of specific structural or potential regulatory domains within these fragments. Furthermore, in case of constructs harboring antisense RNAs of similar length, more efficient were usually those comprising the RBS (ribosome-binding sequence) sites. Such effect was believed to be due to the fact that the antisense RBS sequences prevented gene translation by impeding efficient loading of ribosomes onto phage mRNAs [72]. Overall, expression of phage antisense RNAs in *S. thermophilus* was shown to interfere/delay the intracellular phage DNA replication, decrease phage plaque formation (EOP, efficiency of plating), lower the abundance of phage sense mRNA transcripts and reduce phage progeny particles released from infected cells [73,75].

Similar systems were also developed in *Lactococcus lactis* by expressing anti-sense RNAs directed against various phage genes (e.g. P335-type *gp18C* and *gp24C, gp15C* alone, or putative replication genes, 936-type phage F4-1 major coat protein (*mcp*) gene) [76-79]. In these cases, similarly as for *S. thermophilus* systems, the most efficient antisense RNAs in inhibiting phage development were those comprising the RBS site.

Current data allow to conclude that the most effective antiRNA-based phage defense systems, apart from some exceptions, are those which target: (i) genes vital for phage development (e.g. involved in synthesis of phage DNA), (ii) preferably early-expressed phage genes, (iii) genes expressed at low levels, (iv) genes which respective transcripts are unstable [73,79]. Sequencing of novel phage genomes and development of comparative genomics allows identification of other conserved phage genome regions that could serve as potential targets of antiRNAs.

### *6.6.2. Origin-derived phage-encoded resistance*

Defense systems that employ elements derived from lytic phage genomes are termed phageencoded resistance (PER). One type of engineered PER systems is based on the origin (*ori*) of

phage replication [71]. The principle of such systems relies on presenting *in trans* false targets (in this case, phage-derived *ori*s), which titrate phage replication factors and make them inaccessible for the phage. In result, phage development is inhibited due to arrested replication of its DNA. These engineered systems resemble the naturally-occurring abortive infection mechanisms as they exploit the same principle (for details see: 6.4. Phage abortive infection systems).

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

During the lysogenic life cycle of temperate phages, the lytic module is inactive due to the activity of the CI repressor. However, certain prophage genes - the superinfection-immunity (CI-like repressor) gene itself and the superinfection-exclusion gene, are actively expressed. Both functions were determined to provide protection to the lysogenic host against phage superinfection. Application of these genes to create engineered phage defense systems is yet another strategy of protecting bacterial cells from incoming infections. Multiple bacterial genomes carry prophage-derived sequences, which can count up to 10% of the total genomic content of the cell. Therefore, despite the fact that phage-related sequences are a burden for bacterial cells, they are also believed to provide some advantage to the host by increasing its

Genomic studies in *S. thermophilus* led to the identification of superinfection-immunity (*orf127*) and superinfection-exclusion (*orf203*) genes from the lysogeny module of the Sfi21 prophage [84-85]. Expression of *S. thermophilus* phage Sfi21 *orf127* gene from a plasmid vector conferred the phage resistance phenotype against homologous phage, but was ineffective against other heterologous phages [86]. Analysis of the respective ORF127 product revealed its structural homology with phage λ CI repressor and amino acid homology (15% identity) to a potential CI-like repressor of the lactococcal phage Tuc2009. Gel shift experiments allowed determining the ability of the Sfi21 CI-like repressor to bind to two operator sites identified in the genome of the superinfecting homologous phage Sfi21. Superinfection immunity genes (CI repressors) were also identified in phages of other lactic acid bacteria species (e.g. for *Lactococcus* phage TP901-1 and *Lactobacillus* phages A2 and Фadh) [87-89]. Their expression *in trans* was also reported to provide immunity against

In contrast, superinfection exclusion genes are not engaged in maintaining the lysogenic state, yet are also active during the lysogenic cycle. Experiments based on expression of the *S. thermophilus* phage Sfi21 *orf203* gene *in trans* in high copies determined that it confers resistance to superinfection of a range of heterologous lytic streptococcal phages [85]. Contrarily to the Sfi-21-derived superinfection immunity, in this case resistance to the Sfi21 phage itself was not observed. Moreover, the mechanistic background of the *orf203* dependent resistance phenotype was shown to involve inhibition of phage DNA injection.

A superinfection exclusion system was also developed in *Lactococcus lactis* based on the *sie*2009 gene from the temperate phage Tuc2009 [30]. When cloned *in trans*, *sie*2009 provided resistance only to some 936-type phages used in the study. Moreover, neither c2- nor P335 type phages were affected. It was determined that Sie2009 is a cell membrane-associated protein interfering at the stage of phage DNA injection. However, the exact mechanism by which Sie2009 acts was not yet established. The ability of the designed system to confer resistance only to certain 936-type phages might indicate different mechanisms of DNA injection exhibited by various phages. A similar membrane protein was detected for *S. thermophilus* phage TP-J34, Ltp. Expression of the *ltp* gene provided protection against TP-J34 in *S. thermophilus* and, interestingly in *L. lactis* against a 936-type phage, P008 [32]. This,

*6.6.3. Superinfection immunity and exclusion* 

fitness.

homologous phage infection.

to Lower Phage Contamination in Dairy Fermentation 39

One of the first phage origin-derived systems developed was for *Lactococcus lactis* and employed the *ori* of replication of an industrial phage Φ50 (*ori50*) [80]. Introduction of the Φ50 *ori* region on a high copy number plasmid into the *L. lactis* NCK203 strain provided resistance to not only to phage Φ50 itself, but also to other small isometric phage isolates from industrial environments [80-81]. It was suggested that all of these sensitive phages are part of the same family and most probably exhibit significant homologies within their *ori* regions. Additionally, replication of the *ori50*+ plasmid was shown to be stimulated by Φ50 infection, implying that phage factors are engaged in the process [80]. Further studies determined that the system affects neither adsorption nor phage DNA injection, which suggested that this defense mechanism acts at a later stage of phage development, i.e. DNA replication. It was also clear from the study that the origin-derived phage-encoded resistance phenotype was strongly dependent on the plasmid copy number. Most probably, low copy number plasmids are insufficient in providing enough phage *ori* sites that could efficiently titrate and attract phage replication factors. Yet, on the other hand, when the copy number of *ori*+ plasmids exceeded a certain level, resistant phage mutants were observed as a side-effect. Characterization of these mutants by DNA restriction analysis revealed mutations within the *ori* region, which enabled them to escape the phage defense system.

More recently, a similar origin-derived phage-encoded resistance system was developed for *S. thermophilus* strain Sfi1 based on the *ori* of phage Sfi21 [82]. The presence of this nonencoding phage DNA fragment rendered the Sfi1 host strain resistant to the concomitant phage infection by Sfi21 and 17 other *S. thermophilus* phages. Interestingly, all of them were found to exhibit homology within the *ori* region. However, resistant phages that could overcome this defense mechanism were also detected. They, on the other hand, exhibited differences in the *ori* sequence compared to the wild-type Sfi21-like *ori*. Examination of other *S. thermophilus* phage genomes (~ 30) allowed identifying other distinct replication *ori*s and to divide them into separate groups: replication group I, IIA and IIB [83]. Plasmid constructs harboring these three phage *ori* types increased phage resistance in certain host backgrounds. However, in some strains this origin-derived phage-encoded resistance was not observed. It is therefore speculated whether the efficiency of these systems could be also dependent on some still undetermined host factors.

Development of analogous systems for other lactic acid bacteria involves identification and functional characterization of *ori* regions of their respective phages. This approach can be especially useful for phage-sensitive strains for which other plasmid-encoded defense systems have not yet been determined.

### *6.6.3. Superinfection immunity and exclusion*

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

infection systems).

phage defense system.

dependent on some still undetermined host factors.

systems have not yet been determined.

phage replication [71]. The principle of such systems relies on presenting *in trans* false targets (in this case, phage-derived *ori*s), which titrate phage replication factors and make them inaccessible for the phage. In result, phage development is inhibited due to arrested replication of its DNA. These engineered systems resemble the naturally-occurring abortive infection mechanisms as they exploit the same principle (for details see: 6.4. Phage abortive

One of the first phage origin-derived systems developed was for *Lactococcus lactis* and employed the *ori* of replication of an industrial phage Φ50 (*ori50*) [80]. Introduction of the Φ50 *ori* region on a high copy number plasmid into the *L. lactis* NCK203 strain provided resistance to not only to phage Φ50 itself, but also to other small isometric phage isolates from industrial environments [80-81]. It was suggested that all of these sensitive phages are part of the same family and most probably exhibit significant homologies within their *ori* regions. Additionally, replication of the *ori50*+ plasmid was shown to be stimulated by Φ50 infection, implying that phage factors are engaged in the process [80]. Further studies determined that the system affects neither adsorption nor phage DNA injection, which suggested that this defense mechanism acts at a later stage of phage development, i.e. DNA replication. It was also clear from the study that the origin-derived phage-encoded resistance phenotype was strongly dependent on the plasmid copy number. Most probably, low copy number plasmids are insufficient in providing enough phage *ori* sites that could efficiently titrate and attract phage replication factors. Yet, on the other hand, when the copy number of *ori*+ plasmids exceeded a certain level, resistant phage mutants were observed as a side-effect. Characterization of these mutants by DNA restriction analysis revealed mutations within the *ori* region, which enabled them to escape the

More recently, a similar origin-derived phage-encoded resistance system was developed for *S. thermophilus* strain Sfi1 based on the *ori* of phage Sfi21 [82]. The presence of this nonencoding phage DNA fragment rendered the Sfi1 host strain resistant to the concomitant phage infection by Sfi21 and 17 other *S. thermophilus* phages. Interestingly, all of them were found to exhibit homology within the *ori* region. However, resistant phages that could overcome this defense mechanism were also detected. They, on the other hand, exhibited differences in the *ori* sequence compared to the wild-type Sfi21-like *ori*. Examination of other *S. thermophilus* phage genomes (~ 30) allowed identifying other distinct replication *ori*s and to divide them into separate groups: replication group I, IIA and IIB [83]. Plasmid constructs harboring these three phage *ori* types increased phage resistance in certain host backgrounds. However, in some strains this origin-derived phage-encoded resistance was not observed. It is therefore speculated whether the efficiency of these systems could be also

Development of analogous systems for other lactic acid bacteria involves identification and functional characterization of *ori* regions of their respective phages. This approach can be especially useful for phage-sensitive strains for which other plasmid-encoded defense During the lysogenic life cycle of temperate phages, the lytic module is inactive due to the activity of the CI repressor. However, certain prophage genes - the superinfection-immunity (CI-like repressor) gene itself and the superinfection-exclusion gene, are actively expressed. Both functions were determined to provide protection to the lysogenic host against phage superinfection. Application of these genes to create engineered phage defense systems is yet another strategy of protecting bacterial cells from incoming infections. Multiple bacterial genomes carry prophage-derived sequences, which can count up to 10% of the total genomic content of the cell. Therefore, despite the fact that phage-related sequences are a burden for bacterial cells, they are also believed to provide some advantage to the host by increasing its fitness.

Genomic studies in *S. thermophilus* led to the identification of superinfection-immunity (*orf127*) and superinfection-exclusion (*orf203*) genes from the lysogeny module of the Sfi21 prophage [84-85]. Expression of *S. thermophilus* phage Sfi21 *orf127* gene from a plasmid vector conferred the phage resistance phenotype against homologous phage, but was ineffective against other heterologous phages [86]. Analysis of the respective ORF127 product revealed its structural homology with phage λ CI repressor and amino acid homology (15% identity) to a potential CI-like repressor of the lactococcal phage Tuc2009. Gel shift experiments allowed determining the ability of the Sfi21 CI-like repressor to bind to two operator sites identified in the genome of the superinfecting homologous phage Sfi21. Superinfection immunity genes (CI repressors) were also identified in phages of other lactic acid bacteria species (e.g. for *Lactococcus* phage TP901-1 and *Lactobacillus* phages A2 and Фadh) [87-89]. Their expression *in trans* was also reported to provide immunity against homologous phage infection.

In contrast, superinfection exclusion genes are not engaged in maintaining the lysogenic state, yet are also active during the lysogenic cycle. Experiments based on expression of the *S. thermophilus* phage Sfi21 *orf203* gene *in trans* in high copies determined that it confers resistance to superinfection of a range of heterologous lytic streptococcal phages [85]. Contrarily to the Sfi-21-derived superinfection immunity, in this case resistance to the Sfi21 phage itself was not observed. Moreover, the mechanistic background of the *orf203* dependent resistance phenotype was shown to involve inhibition of phage DNA injection.

A superinfection exclusion system was also developed in *Lactococcus lactis* based on the *sie*2009 gene from the temperate phage Tuc2009 [30]. When cloned *in trans*, *sie*2009 provided resistance only to some 936-type phages used in the study. Moreover, neither c2- nor P335 type phages were affected. It was determined that Sie2009 is a cell membrane-associated protein interfering at the stage of phage DNA injection. However, the exact mechanism by which Sie2009 acts was not yet established. The ability of the designed system to confer resistance only to certain 936-type phages might indicate different mechanisms of DNA injection exhibited by various phages. A similar membrane protein was detected for *S. thermophilus* phage TP-J34, Ltp. Expression of the *ltp* gene provided protection against TP-J34 in *S. thermophilus* and, interestingly in *L. lactis* against a 936-type phage, P008 [32]. This,

quite surprising observation of *sie*-encoded cross-resistance was argued to be due to a recent genetic transfer event between the two species. In both cases, it was noted that phage adsorption was not impaired, but there was significant inhibition of phage DNA accumulation within the host cell. Based on these observations it was proposed that the *ltp* gene product acts at the stage of phage DNA injection by either impairing insertion of the phage tail into the cytoplasmic membrane or by obscuring the host membrane protein responsible for inducing the release of phage DNA from the capsid.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

the phage Ф31 middle-expressed promoter (Ф31*p*) that is active at a significant level solely after Ф31 infection [91]. Expression of this plasmid-encoded suicide system was designed to restrict unmethylated host and phage DNA upon infection. During infection, induction of the *llaIR+* cassette caused a significant drop of phage Ф31 EOP. Only a small fraction of infected cells produced progeny phage particles. The system provided also protection against other Ф31-like phages. Yet, despite the observed inhibition of phage development, some phages were found to escape this defense system. Phage mutants that emerged during the assays were all found to be altered in the sequence encoding the transcriptional activator of the Ф31*p* promoter [92]. Thus, these mutants escaped the system due to lack of efficient transcription of the *llaIR+* cassette. The drawback of this system is the fact that it is only active against phages that can trigger the phage-derived promoter; in this case, against Ф31 and its closely related phages. Another disadvantage is the fact that the *llaIR+* cassette is not expressed immediately, but after a time necessary for the infecting phage to synthesize transcription factors activating the middle-expressed promoter. This, in effect, allows for replication of a low number of phage particles that escape restriction. It would seem more appropriate to use early phage promoters; yet, these are usually host-controlled. Improvement of the efficiency of the already existing suicide systems should involve cautious selection of effectively controlled and adequately strong phage promoters and more proficient restriction endonucleases. This is best illustrated by the described earlier observation that application of a stronger promoter, despite a more efficient reduction of phage EOP, can have a negative effect on bacterial cell growth. As a method to enhance the efficacy of the suicide system, it was proposed to include within the suicide cassette another gene - *llaIC*, encoding a regulator protein [93]. The presence of this regulator protein was

suggested to significantly increase the anti-phage restriction activity of LlaIR.

other protein components, away from the wild-type proteins.

The subunit poisoning system is an engineered phage defense strategy that relies on expression *in trans* of truncated/mutated proteins which impair (poison) the function of their wild type variants. To achieve this, mutant proteins should be expressed at levels higher than their wild-type counterparts. Moreover, despite alterations in their amino acid sequences, they must have an intact structural form in order to titrate sites or substrates, or

An example of such system is based on the CI-like repressor of lytic *Lactococcus lactis* phage Ф31. The general idea of this strategy resembles very much the superinfection immunity approach, where *S. thermophilus* bacteria expressing the phage Sfi21 CI repressor were protected from closely-related phages (for details see: *6.6.3.* Superinfection immunity and exclusion). However, in this case, the exact mechanism is somewhat different [94]. Studies of the wild type CI repressor of phage Ф31 showed that it is non-functional and, when expressed in the *L. lactis* host, does not provide protection against superinfecting phages nor represses the transcription of phage lytic genes. Yet, when this wild-type Ф31 CI protein or its truncated variants were expressed *in trans*, they could efficiently inhibit infection of Ф31

*6.6.5. Subunit poisoning* 

to Lower Phage Contamination in Dairy Fermentation 41

Putative superinfection exclusion genes seem to be widespread among prophage-containing lactococcal and streptococcal strains and localized in the same genomic region limited by repressor and integrase gene from each side. Although *sie* genes lack significant homology, all currently identified Sie proteins are small with hydrophobic N' tail and at least one transmembrane domain. Various studies of lactococcal Sie proteins allowed grouping them into several phylogenetic groups, depending on the subset of 936-type phages they target (sk1/jj50, bIL170/p008 or 712 group) [28]. At present, it is argued that all *sie* systems identified for lactococcal lysogenic strains interact or mask cell membrane associated factors engaged in phage DNA injection or come into direct contact with structural proteins of the infecting phage. The most probable theory is that Sie function is aimed against the tail tape measure protein (function implicated in phage DNA injection process), as its encoding region is among the few divergent genomic regions between the different subsets of 936 type phages.

Superinfection exclusion and immunity genes in natural conditions can also be provided by defective prophages. The nature of defective phages is that they cannot be efficiently induced by environmental factors; hence, cured from the host strain. Such lysogenic lactic acid bacterial strains (particularly *Lactobacillus* species), which exhibit no threat to the fermentation processes due to uncontrolled prophage induction, are of special interest to the dairy industry as naturally-resistant strains to superinfection events.

### *6.6.4. Phage-triggered suicide systems*

Phage-triggered suicide systems rely on expression of toxic elements under the strict control of phage-inducible promoters. Such specifically engineered systems most closely resemble the naturally-occurring abortive infection systems, which trap the phage within infected cells and lead to programmed cell death. Upon phage infection, host cells are lysed, disabling at the same time phage propagation and the concomitant spread of the phage. In effect, the uninfected bacterial population is saved (for details see: 6.4. Phage abortive infection systems). Suicide systems are based on three genetic components: (i) a lethal gene cassette, (ii) a phage promoter induced only after phage infection, and (iii) an appropriate vector, providing sufficient amount of the lethal gene product.

Such system, based on an inducible plasmid strategy, was created for *L. lactis* to control phage infections [90]. The system comprises a lethal three gene cassette, *llaIR+*, encoding a restriction endonuclease of the *L. lactis* LlaI R/M system, cloned under the tight control of the phage Ф31 middle-expressed promoter (Ф31*p*) that is active at a significant level solely after Ф31 infection [91]. Expression of this plasmid-encoded suicide system was designed to restrict unmethylated host and phage DNA upon infection. During infection, induction of the *llaIR+* cassette caused a significant drop of phage Ф31 EOP. Only a small fraction of infected cells produced progeny phage particles. The system provided also protection against other Ф31-like phages. Yet, despite the observed inhibition of phage development, some phages were found to escape this defense system. Phage mutants that emerged during the assays were all found to be altered in the sequence encoding the transcriptional activator of the Ф31*p* promoter [92]. Thus, these mutants escaped the system due to lack of efficient transcription of the *llaIR+* cassette. The drawback of this system is the fact that it is only active against phages that can trigger the phage-derived promoter; in this case, against Ф31 and its closely related phages. Another disadvantage is the fact that the *llaIR+* cassette is not expressed immediately, but after a time necessary for the infecting phage to synthesize transcription factors activating the middle-expressed promoter. This, in effect, allows for replication of a low number of phage particles that escape restriction. It would seem more appropriate to use early phage promoters; yet, these are usually host-controlled. Improvement of the efficiency of the already existing suicide systems should involve cautious selection of effectively controlled and adequately strong phage promoters and more proficient restriction endonucleases. This is best illustrated by the described earlier observation that application of a stronger promoter, despite a more efficient reduction of phage EOP, can have a negative effect on bacterial cell growth. As a method to enhance the efficacy of the suicide system, it was proposed to include within the suicide cassette another gene - *llaIC*, encoding a regulator protein [93]. The presence of this regulator protein was suggested to significantly increase the anti-phage restriction activity of LlaIR.

### *6.6.5. Subunit poisoning*

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

type phages.

responsible for inducing the release of phage DNA from the capsid.

dairy industry as naturally-resistant strains to superinfection events.

vector, providing sufficient amount of the lethal gene product.

*6.6.4. Phage-triggered suicide systems* 

quite surprising observation of *sie*-encoded cross-resistance was argued to be due to a recent genetic transfer event between the two species. In both cases, it was noted that phage adsorption was not impaired, but there was significant inhibition of phage DNA accumulation within the host cell. Based on these observations it was proposed that the *ltp* gene product acts at the stage of phage DNA injection by either impairing insertion of the phage tail into the cytoplasmic membrane or by obscuring the host membrane protein

Putative superinfection exclusion genes seem to be widespread among prophage-containing lactococcal and streptococcal strains and localized in the same genomic region limited by repressor and integrase gene from each side. Although *sie* genes lack significant homology, all currently identified Sie proteins are small with hydrophobic N' tail and at least one transmembrane domain. Various studies of lactococcal Sie proteins allowed grouping them into several phylogenetic groups, depending on the subset of 936-type phages they target (sk1/jj50, bIL170/p008 or 712 group) [28]. At present, it is argued that all *sie* systems identified for lactococcal lysogenic strains interact or mask cell membrane associated factors engaged in phage DNA injection or come into direct contact with structural proteins of the infecting phage. The most probable theory is that Sie function is aimed against the tail tape measure protein (function implicated in phage DNA injection process), as its encoding region is among the few divergent genomic regions between the different subsets of 936-

Superinfection exclusion and immunity genes in natural conditions can also be provided by defective prophages. The nature of defective phages is that they cannot be efficiently induced by environmental factors; hence, cured from the host strain. Such lysogenic lactic acid bacterial strains (particularly *Lactobacillus* species), which exhibit no threat to the fermentation processes due to uncontrolled prophage induction, are of special interest to the

Phage-triggered suicide systems rely on expression of toxic elements under the strict control of phage-inducible promoters. Such specifically engineered systems most closely resemble the naturally-occurring abortive infection systems, which trap the phage within infected cells and lead to programmed cell death. Upon phage infection, host cells are lysed, disabling at the same time phage propagation and the concomitant spread of the phage. In effect, the uninfected bacterial population is saved (for details see: 6.4. Phage abortive infection systems). Suicide systems are based on three genetic components: (i) a lethal gene cassette, (ii) a phage promoter induced only after phage infection, and (iii) an appropriate

Such system, based on an inducible plasmid strategy, was created for *L. lactis* to control phage infections [90]. The system comprises a lethal three gene cassette, *llaIR+*, encoding a restriction endonuclease of the *L. lactis* LlaI R/M system, cloned under the tight control of The subunit poisoning system is an engineered phage defense strategy that relies on expression *in trans* of truncated/mutated proteins which impair (poison) the function of their wild type variants. To achieve this, mutant proteins should be expressed at levels higher than their wild-type counterparts. Moreover, despite alterations in their amino acid sequences, they must have an intact structural form in order to titrate sites or substrates, or other protein components, away from the wild-type proteins.

An example of such system is based on the CI-like repressor of lytic *Lactococcus lactis* phage Ф31. The general idea of this strategy resembles very much the superinfection immunity approach, where *S. thermophilus* bacteria expressing the phage Sfi21 CI repressor were protected from closely-related phages (for details see: *6.6.3.* Superinfection immunity and exclusion). However, in this case, the exact mechanism is somewhat different [94]. Studies of the wild type CI repressor of phage Ф31 showed that it is non-functional and, when expressed in the *L. lactis* host, does not provide protection against superinfecting phages nor represses the transcription of phage lytic genes. Yet, when this wild-type Ф31 CI protein or its truncated variants were expressed *in trans*, they could efficiently inhibit infection of Ф31

and other lytic P335-type phages. Expression of Ф31-derived *c*I mutant genes from a highcopy number vector was shown to inhibit growth of Ф31 (to EOP 10-6 or lower, depending on the mutation) and of other lytic P335-type phages. The observed effect was determined to be due to the competitive binding of the non-functional Ф31 CI and phage-expressed CI repressors to two of the three wild-type operators identified within the genetic switch region. It was suggested that the truncated variants of the Ф31 CI repressor exhibit a higher affinity for these sites than the phage-encoded CI protein. In effect of Ф31 CI binding, expression of lytic phage functions was repressed, impairing phage DNA replication.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

development in the genome of the *S. thermophilus* Sfi strain [96]. The *orf394* gene, encoding a putative transmembrane protein, was one of the host loci determined to confer phage resistance to Sfi19 as well as more than 10 other heterologous *S. thermophilus* phages. After infection by phage Sfi19, no phage DNA synthesis was detected for such mutant strain. Based on this observation, it was suggested that this transmembrane protein may be implicated in the stage of phage DNA injection, analogously to the Pip (phage infection protein) of *Lactococcus lactis* [97]. Among advantages of the host-factor elimination system is the fact that it is food-grade and that the engineered phage resistant strains can be successfully used in production processes. Yet, it must be noted that before its application in the industry, the strain should be assayed for its phage resistance phenotype during several rotation rounds of culturing. There is also no data on the phage mutants that can evolve due

mutant strains.

Other host factors that were suggested to be efficient targets for developing phage defense systems are auxotrophic genes. Pedersen et al. developed a strategy of impairing phage replication in an industrial *Lactococcus lactis* strain by deleting the thymidylate synthase (*thyA*) gene from its genome [98]. This patented strategy is based on the process of phage DNA replication [99]. Upon infection, phages take advantage of the DNA replication machinery of the host to amplify its own genetic material. However, when the host is lacking one of the main DNA building factors, formation of novel replicated DNA molecules is inhibited. The *thyA* gene is responsible for *de novo* synthesis of dTTP in the cell. Strains devoid of *thyA* cannot synthesize dTTP in the medium that lacks thymidine, such as milk. Under such conditions, the Δ*thyA* mutant was resistant to infection by selected P335- and 936-type phages, which efficiently infected the parental wild type strain, and what is important its acidifying properties remained undisturbed. Addition of thymidine to the milk medium restored phage sensitivity of the strain. Among the drawbacks of this system is the fact that the mutant strain lacking thymidylate synthase is impaired for growth. Therefore, in industrial conditions it must be inoculated into milk tanks at higher concentrations than the parental wild type strain in order to meet the technological criteria. A solution to this problem could be addition of limiting amounts of thymidine to promote growth of such starter strain. Yet, thymidine as an additive in the cheese industry is not allowed. Among various options proposed by Pedersen to obtain a phage-resistant *thy* mutant for industrial use could be construction of a thermosensitive mutant, in which expression of the *thy* gene is inhibited at temperatures at which technological processes are carried out [98]. The greatest concern when selecting for phage-resistant strains is their ability to prevail a broad range of phages over long periods of time. In this case, it seems that the host-factor elimination system is the most universal among the presented systems as it acts against all phage types. Moreover, at this point it seems that the probability of occurrence of phage mutants overcoming this resistance mechanism is low. However, it should also be noted that some phages are known to encode own *thy* genes or utilize nucleotides of the host by hydrolyzing its DNA, which

to the continuous use of such Pip-

can be a weak point [100-101].

to Lower Phage Contamination in Dairy Fermentation 43

During the study resistant phages were also detected. Sequence analysis studies within the genetic switch regions revealed alterations in their operator sites, which impaired binding of the Ф31-derived CI repressor.

Another example of subunit poisoning phage defense is a system developed in *Streptococcus thermophilus*. The strategy was based on mutating the primase-encoding gene, an essential component of the replication module of the *S. thermophilus* Sfi-21-like 3 phage [95]. Mutation of sequences within the highly conserved domains of the primase gene resulted in obtaining dysfunctional protein variants. Expression at high level of such primase derivatives *in trans* resulted in reduction of EOP of 3 and several other Sfi-21-type phages due to inhibition of phage DNA replication. The mutated primase was implied to titrate replication factors and/or the origin of replication, making them inaccessible for the wildtype primase. This suggestion seems to be credible as introduction of the STOP codon upstream of the initial gene mutations restored the phage sensitivity phenotype. Such alteration lead to the synthesis of a truncated protein, which, most probably, could no longer mimic the structure of the native primase. A great advantage of this primase-based subunit poisoning system is lack of phage mutants resistant to the mutated primase proteins under study.

Overall, subunit poisoning is an approach that is believed to constitute a broad phage defense system, as it was shown to be effective against more than one lytic P335-type phage. In this aspect, it differs from the earlier described superinfection immunity systems, where expression of phage repressor genes from phages of various lactic acid bacterial species (e.g. *Streptococcus* phage Sfi21, *Lactococcus* phage TP901-1 or *Lactobacillus* A2 or Фadh phages) provided immunity against the respective single phage only [85,87-89].

### *6.6.6. Host-factor elimination*

Eliminating a genetic element from the genome of starter bacteria to obtain phage-resistant strains is yet another strategy of engineering a phage defense system. This approach can target different stages of the phage life cycle, which are often host-dependent, e.g. phage injection dependent on host membrane proteins, host factors necessary for phage DNA replication.

Among methods identifying such host-encoded factors is random mutagenesis using the pGhost::IS*S1* mutation vector. This approach allowed to identify genes necessary for phage development in the genome of the *S. thermophilus* Sfi strain [96]. The *orf394* gene, encoding a putative transmembrane protein, was one of the host loci determined to confer phage resistance to Sfi19 as well as more than 10 other heterologous *S. thermophilus* phages. After infection by phage Sfi19, no phage DNA synthesis was detected for such mutant strain. Based on this observation, it was suggested that this transmembrane protein may be implicated in the stage of phage DNA injection, analogously to the Pip (phage infection protein) of *Lactococcus lactis* [97]. Among advantages of the host-factor elimination system is the fact that it is food-grade and that the engineered phage resistant strains can be successfully used in production processes. Yet, it must be noted that before its application in the industry, the strain should be assayed for its phage resistance phenotype during several rotation rounds of culturing. There is also no data on the phage mutants that can evolve due to the continuous use of such Pip mutant strains.

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

the Ф31-derived CI repressor.

under study.

replication.

*6.6.6. Host-factor elimination* 

and other lytic P335-type phages. Expression of Ф31-derived *c*I mutant genes from a highcopy number vector was shown to inhibit growth of Ф31 (to EOP 10-6 or lower, depending on the mutation) and of other lytic P335-type phages. The observed effect was determined to be due to the competitive binding of the non-functional Ф31 CI and phage-expressed CI repressors to two of the three wild-type operators identified within the genetic switch region. It was suggested that the truncated variants of the Ф31 CI repressor exhibit a higher affinity for these sites than the phage-encoded CI protein. In effect of Ф31 CI binding,

expression of lytic phage functions was repressed, impairing phage DNA replication.

During the study resistant phages were also detected. Sequence analysis studies within the genetic switch regions revealed alterations in their operator sites, which impaired binding of

Another example of subunit poisoning phage defense is a system developed in *Streptococcus thermophilus*. The strategy was based on mutating the primase-encoding gene, an essential component of the replication module of the *S. thermophilus* Sfi-21-like 3 phage [95]. Mutation of sequences within the highly conserved domains of the primase gene resulted in obtaining dysfunctional protein variants. Expression at high level of such primase derivatives *in trans* resulted in reduction of EOP of 3 and several other Sfi-21-type phages due to inhibition of phage DNA replication. The mutated primase was implied to titrate replication factors and/or the origin of replication, making them inaccessible for the wildtype primase. This suggestion seems to be credible as introduction of the STOP codon upstream of the initial gene mutations restored the phage sensitivity phenotype. Such alteration lead to the synthesis of a truncated protein, which, most probably, could no longer mimic the structure of the native primase. A great advantage of this primase-based subunit poisoning system is lack of phage mutants resistant to the mutated primase proteins

Overall, subunit poisoning is an approach that is believed to constitute a broad phage defense system, as it was shown to be effective against more than one lytic P335-type phage. In this aspect, it differs from the earlier described superinfection immunity systems, where expression of phage repressor genes from phages of various lactic acid bacterial species (e.g. *Streptococcus* phage Sfi21, *Lactococcus* phage TP901-1 or *Lactobacillus* A2 or Фadh phages)

Eliminating a genetic element from the genome of starter bacteria to obtain phage-resistant strains is yet another strategy of engineering a phage defense system. This approach can target different stages of the phage life cycle, which are often host-dependent, e.g. phage injection dependent on host membrane proteins, host factors necessary for phage DNA

Among methods identifying such host-encoded factors is random mutagenesis using the pGhost::IS*S1* mutation vector. This approach allowed to identify genes necessary for phage

provided immunity against the respective single phage only [85,87-89].

Other host factors that were suggested to be efficient targets for developing phage defense systems are auxotrophic genes. Pedersen et al. developed a strategy of impairing phage replication in an industrial *Lactococcus lactis* strain by deleting the thymidylate synthase (*thyA*) gene from its genome [98]. This patented strategy is based on the process of phage DNA replication [99]. Upon infection, phages take advantage of the DNA replication machinery of the host to amplify its own genetic material. However, when the host is lacking one of the main DNA building factors, formation of novel replicated DNA molecules is inhibited. The *thyA* gene is responsible for *de novo* synthesis of dTTP in the cell. Strains devoid of *thyA* cannot synthesize dTTP in the medium that lacks thymidine, such as milk. Under such conditions, the Δ*thyA* mutant was resistant to infection by selected P335- and 936-type phages, which efficiently infected the parental wild type strain, and what is important its acidifying properties remained undisturbed. Addition of thymidine to the milk medium restored phage sensitivity of the strain. Among the drawbacks of this system is the fact that the mutant strain lacking thymidylate synthase is impaired for growth. Therefore, in industrial conditions it must be inoculated into milk tanks at higher concentrations than the parental wild type strain in order to meet the technological criteria. A solution to this problem could be addition of limiting amounts of thymidine to promote growth of such starter strain. Yet, thymidine as an additive in the cheese industry is not allowed. Among various options proposed by Pedersen to obtain a phage-resistant *thy* mutant for industrial use could be construction of a thermosensitive mutant, in which expression of the *thy* gene is inhibited at temperatures at which technological processes are carried out [98]. The greatest concern when selecting for phage-resistant strains is their ability to prevail a broad range of phages over long periods of time. In this case, it seems that the host-factor elimination system is the most universal among the presented systems as it acts against all phage types. Moreover, at this point it seems that the probability of occurrence of phage mutants overcoming this resistance mechanism is low. However, it should also be noted that some phages are known to encode own *thy* genes or utilize nucleotides of the host by hydrolyzing its DNA, which can be a weak point [100-101].
