**12.2. BIM system - exposure of sensitive strains to lytic phages (spontaneous mutation in chromosomal or plasmidic genes)**

Selection of BIMs (bacteriophage insensitive mutants) is a way to obtain phage-resistant strains without genetic manipulations. The idea of obtaining such cells is to infect a starter strain culture and select for mutants which have sustained phage attack.

This approach has its drawbacks, as it is based solely on the occurrence of random potential mutations in genes coding for receptor materials. The lack of a functional initial receptor for 936- and P335-type phages, such as a polysaccharide, is associated with mutations in genes involved in its synthesis or transport. It is well documented that phage insensitivity of *L. lactis* strains is correlated with loss of the galactose-associated receptor in the cell wall. This disturbs the synthesis of wall components and, as a consequence, insensitive strains often lose their industrial properties, such as the ability to produce acids, and reveal weaker growth in comparison to wild type strains.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

mechanisms to plasmid molecules, were simple assays based on isolation of plasmids from resistant strains and their reintroduction into susceptible cells to obtain cells immune to attack by a particular phage. The later discovery of phage resistance determinants encoded on conjugational plasmids attracted great interest of the food production industry. Most of the data on conjugative plasmids conferring phage resistance comes from studies in *Lactococcus lactis*. In this species many various conjugal plasmids conferring phageresistance have been identified, including: pTN20, pNP40 and pCI1750, carrying both conjugal transfer (Tra+) and abortive infection (Abi+) determinants, or pAJ1106, exhibiting Tra+ and Hsp+ phenotype [145-149]. Extensive studies of various research groups showed that indeed construction of phage-resistant strains via simple conjugational transfer is an effective means of generating phage resistant starter strains, some of which found

Among the first conjugal plasmids discovered in *Lactococcus lactis* was pTR2030 isolated from strain ME2. It was characterized to encode heat-sensitive phage resistance (Hsp+), restriction-modification (LlaIR/M) as well as conjugal transfer (Tra+) genes [151]. Its introduction via conjugation into other lactococcal strains, including *Lactococcus lactis* subsp*. cremoris*, resulted in phage-resistance phenotypes [152]. Application of these genetic elements was hence proclaimed as an attractive and acceptable alternative for generating resistant strains, in contrast to strain construction using genetic engineering. The study of Sanders et al. (1986) described the successful attempt of introducing the pTR2030 plasmid via conjugation from a *L. lactis* donor into several industrial recipient strains, from both *lactis* and *cremoris* subspecies [143]. Resulting transconjugants proved resistant to homologous phage infection. Curing of pTR2030 from transconjugants restored phage-sensitive phenotypes, proving visibly that phage resistance is conferred by the plasmid. Noteworthy is the fact that selection of phage-resistant transconjugants was performed in an antibioticfree background, which is most appropriate for manipulations with strains intended for food production. Another important advantage of this approach was the fact that transconjugant strains maintained their acid-producing properties. This aspect is quite important as it shows that conjugative plasmid manipulations do not alter the industrially attractive features of starter bacteria. The pTR2030 plasmid was maintained throughout multiple generations, indicating that phage resistance will be a stable feature during prolonged use of the transconjugant in industrial applications. Resistance mechanisms identified on conjugative plasmids were also applied in developing engineered bacterial phage defense systems, e.g. the LlaIR/M function encoded on the pTR2030 plasmid was

used in constructing phage-triggered suicide systems (see section: *6.6.4.*).

The plasmid-concept of generating phage-resistant strains has also its limitations. First of all, it should be taken into account that many industrially-applied strains are hard to transform. Furthermore, there is a chance that introduction of new plasmids might destabilize industrially attractive strain properties that are also plasmid-encoded (issue of plasmid incompatibility). Introduction of plasmids transferring phage resistance into the bacterial chromosome could be a way of stabilizing this feature; yet, on the other hand, will demand

application in the dairy industry [143,150].

to Lower Phage Contamination in Dairy Fermentation 59

Apart from altering cell growth, other two features, such as narrow phage specificity and spontaneous reversion to sensitive phenotype, limit exploitation of BIM mutants in industrial applications [17]. However, mutations in the *pip* gene, encoding a specific receptor for c2-type phages only (for further details on Pip function see: 4 and 6.1-2.), have no significant impact on vitality of lactococcal cells and resultant mutants are stably maintained [17,24]. Genetic engineering methods, which possess a huge potential for developing protection against phages, based on specific point mutations, and construction of stable mutants, might be the solution to this problem. However, at present methods utilizing recombinant DNA approaches restrict the industrial use of genetically modified strains. Mills and colleagues presented a simple 3-step approach, devoid of genetic engineering methods, for generating BIMs of *S. thermophilus* [141]. In the first step, sensitive bacteria were completely lysed in soft top agar plates by adding a selected industrial phage at a MOI > 1 (multiplicity of infection above 1). Subsequently, plates were incubated up to 48 hours after which appearance of resistant colonies was observed. In the next step, all colonies were collected and used to inoculate fresh liquid medium. Harvested bacteria from step 2 were used for conducting a continuous culture in milk with 20–25 passages in the presence of phage at a high concentration (MOI = 10). In order to obtain BIM colonies, the last passage was poured on solid agar from which phage-resistant BIMs were selected after overnight growth. Resistance to another phage could be generated by repeating the whole process on the resultant BIM strain. The insensitive phenotype was initially attributed to nonspecific mutations in receptor genes. However, further studies revealed that phage insensitivity is due to alteration of the CRISPR (clustered regularly interspaced short palindromic repeats) locus, not associated with the previously thought mutations [142] (for further details on CRISPRs see section 6.5 and 12.4).

### **12.3. Plasmid concept**

Among the acknowledged and widely applied methods of obtaining starter strains resistant to phage infections is conjugational transfer of plasmids conferring phage resistance determinants [143-144]. In lactococci, there is a range of bacteriophage defense systems occurring naturally on plasmids (**natural, plasmid-encoded phage-resistance systems**). Among the plasmid-encoded phage resistance are such defense mechanisms as restriction/modification (R/M) or abortive infection (Hsp+ or other Abi+) (for more details see sections: 6.3. and 6.4.). First studies, which linked the presence of phage resistance mechanisms to plasmid molecules, were simple assays based on isolation of plasmids from resistant strains and their reintroduction into susceptible cells to obtain cells immune to attack by a particular phage. The later discovery of phage resistance determinants encoded on conjugational plasmids attracted great interest of the food production industry. Most of the data on conjugative plasmids conferring phage resistance comes from studies in *Lactococcus lactis*. In this species many various conjugal plasmids conferring phageresistance have been identified, including: pTN20, pNP40 and pCI1750, carrying both conjugal transfer (Tra+) and abortive infection (Abi+) determinants, or pAJ1106, exhibiting Tra+ and Hsp+ phenotype [145-149]. Extensive studies of various research groups showed that indeed construction of phage-resistant strains via simple conjugational transfer is an effective means of generating phage resistant starter strains, some of which found application in the dairy industry [143,150].

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

growth in comparison to wild type strains.

CRISPRs see section 6.5 and 12.4).

**12.3. Plasmid concept** 

This approach has its drawbacks, as it is based solely on the occurrence of random potential mutations in genes coding for receptor materials. The lack of a functional initial receptor for 936- and P335-type phages, such as a polysaccharide, is associated with mutations in genes involved in its synthesis or transport. It is well documented that phage insensitivity of *L. lactis* strains is correlated with loss of the galactose-associated receptor in the cell wall. This disturbs the synthesis of wall components and, as a consequence, insensitive strains often lose their industrial properties, such as the ability to produce acids, and reveal weaker

Apart from altering cell growth, other two features, such as narrow phage specificity and spontaneous reversion to sensitive phenotype, limit exploitation of BIM mutants in industrial applications [17]. However, mutations in the *pip* gene, encoding a specific receptor for c2-type phages only (for further details on Pip function see: 4 and 6.1-2.), have no significant impact on vitality of lactococcal cells and resultant mutants are stably maintained [17,24]. Genetic engineering methods, which possess a huge potential for developing protection against phages, based on specific point mutations, and construction of stable mutants, might be the solution to this problem. However, at present methods utilizing recombinant DNA approaches restrict the industrial use of genetically modified strains. Mills and colleagues presented a simple 3-step approach, devoid of genetic engineering methods, for generating BIMs of *S. thermophilus* [141]. In the first step, sensitive bacteria were completely lysed in soft top agar plates by adding a selected industrial phage at a MOI > 1 (multiplicity of infection above 1). Subsequently, plates were incubated up to 48 hours after which appearance of resistant colonies was observed. In the next step, all colonies were collected and used to inoculate fresh liquid medium. Harvested bacteria from step 2 were used for conducting a continuous culture in milk with 20–25 passages in the presence of phage at a high concentration (MOI = 10). In order to obtain BIM colonies, the last passage was poured on solid agar from which phage-resistant BIMs were selected after overnight growth. Resistance to another phage could be generated by repeating the whole process on the resultant BIM strain. The insensitive phenotype was initially attributed to nonspecific mutations in receptor genes. However, further studies revealed that phage insensitivity is due to alteration of the CRISPR (clustered regularly interspaced short palindromic repeats) locus, not associated with the previously thought mutations [142] (for further details on

Among the acknowledged and widely applied methods of obtaining starter strains resistant to phage infections is conjugational transfer of plasmids conferring phage resistance determinants [143-144]. In lactococci, there is a range of bacteriophage defense systems occurring naturally on plasmids (**natural, plasmid-encoded phage-resistance systems**). Among the plasmid-encoded phage resistance are such defense mechanisms as restriction/modification (R/M) or abortive infection (Hsp+ or other Abi+) (for more details see sections: 6.3. and 6.4.). First studies, which linked the presence of phage resistance Among the first conjugal plasmids discovered in *Lactococcus lactis* was pTR2030 isolated from strain ME2. It was characterized to encode heat-sensitive phage resistance (Hsp+), restriction-modification (LlaIR/M) as well as conjugal transfer (Tra+) genes [151]. Its introduction via conjugation into other lactococcal strains, including *Lactococcus lactis* subsp*. cremoris*, resulted in phage-resistance phenotypes [152]. Application of these genetic elements was hence proclaimed as an attractive and acceptable alternative for generating resistant strains, in contrast to strain construction using genetic engineering. The study of Sanders et al. (1986) described the successful attempt of introducing the pTR2030 plasmid via conjugation from a *L. lactis* donor into several industrial recipient strains, from both *lactis* and *cremoris* subspecies [143]. Resulting transconjugants proved resistant to homologous phage infection. Curing of pTR2030 from transconjugants restored phage-sensitive phenotypes, proving visibly that phage resistance is conferred by the plasmid. Noteworthy is the fact that selection of phage-resistant transconjugants was performed in an antibioticfree background, which is most appropriate for manipulations with strains intended for food production. Another important advantage of this approach was the fact that transconjugant strains maintained their acid-producing properties. This aspect is quite important as it shows that conjugative plasmid manipulations do not alter the industrially attractive features of starter bacteria. The pTR2030 plasmid was maintained throughout multiple generations, indicating that phage resistance will be a stable feature during prolonged use of the transconjugant in industrial applications. Resistance mechanisms identified on conjugative plasmids were also applied in developing engineered bacterial phage defense systems, e.g. the LlaIR/M function encoded on the pTR2030 plasmid was used in constructing phage-triggered suicide systems (see section: *6.6.4.*).

The plasmid-concept of generating phage-resistant strains has also its limitations. First of all, it should be taken into account that many industrially-applied strains are hard to transform. Furthermore, there is a chance that introduction of new plasmids might destabilize industrially attractive strain properties that are also plasmid-encoded (issue of plasmid incompatibility). Introduction of plasmids transferring phage resistance into the bacterial chromosome could be a way of stabilizing this feature; yet, on the other hand, will demand approval of appropriate authorities. Furthermore, some industrially-exploited lactic acid bacteria species, e.g. *S. thermophilus*, carry few plasmids (including conjugal plasmids). This can be an obstacle in generating novel phage-resistant strains via conjugational events [153]. Yet, studies performed by Burrus et al. (2001) revealed the presence of an integrative conjugative element ICR*St1* in *S. thermophilus* strain CNRZ368, shown to encode a II-type R/M system that provided resistance to phage φST84 infection [154]. Identification of a phage defense system on an integrative element suggests that also such genetic elements as transposons can be responsible for the spread of phage-resistance mechanisms within bacterial populations.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

spontaneous *S. thermophilus* BIM cells by providing selection pressure due to phage infection. Protocols of isolating such strains have been later developed for dairy *S. thermophilus*, applied in the manufacturing of cheese and yoghurts [141]. The strategy is based on exposition of bacterial starter culture to high phage titers. Several rounds of growth in milk media under the constant selection pressure due to the phage presence resulted in obtaining phage-resistant mutants able to efficiently grow under industrial conditions. The great advantage of such approach is the fact that the presence of naturally acquired spacer sequences renders the strain resistant to phage infections, while preserving the industrially-attractive features of the initial starter cultures. Another strategy of constructing phage-resistant strains could be deliberate integration of synthetic spacers homologous to conserved sequences of industrial phage isolates into the CRISPR array of starter bacteria. However, this approach would involve certain molecular manipulations at the DNA level. Nonetheless, controlled modification of phage resistance of LAB strains using the CRISPR/*cas* regions is not considered by the food industry as a genetic

modification method within the meaning of the existing rules in this area.

*Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Warsaw, Poland* 

[1] Whitehead HR, Cox GA. The occurrence of bacteriophage in lactic streptococci. N. Z. J.

[2] Daly C, Fitzgerald GF, Davis R. Biotechnology of lactic acid bacteria with special reference to bacteriophage resistance. Antonie Van Leeuwenhoek 1996;70(2-4) 99-110. [3] Lawrence R C. Action of bacteriophages on lactic acid bacteria: consequences and

[6] Klaenhammer T, Altermann E, Arigoni F, Bolotin A, Breidt F, Broadbent J, Cano R, Chaillou S, Deutscher J, Gasson M, van de Guchte M, Guzzo J, Hartke A, Hawkins T, Hols P, Hutkins R, Kleerebezem M, Kok J, Kuipers O, Lubbers M, Maguin E, McKay L, Mills D, Nauta A, Overbeek R, Pel H, Pridmore D, Saier M, van Sinderen D, Sorokin A, Steele J, O'Sullivan D, de Vos W, Weimer B, Zagorec M, Siezen R. Discovering lactic

[4] Brüssow H, Hendrix RW. Phage genomics: small is beautiful. Cell 2002;108(1) 13-6. [5] Kołakowski P, Rybka J. Causes of disorders of fermentation failures (in Polish). BIBIT

acid bacteria by genomics. Antonie Van Leeuwenhoek 2002;82(1-4) 29-58.

A.K. Szczepankowska, R.K. Górecki and J.K. Bardowski

protection. N. Z. J. Dairy Sci. Technol. 1978;13 129-136.

(Information Bulletin Rhodia Food Biolacta) 2001;2(24) 7-14.

*Danisco Biolacta, Innovation, Olsztyn, Poland* 

Dairy Sci. Technol. 1935;16 319–320.

**Author details** 

P. Kołakowski \*

**13. References** 

 \*

Corresponding Author

to Lower Phage Contamination in Dairy Fermentation 61

### **12.4. CRISPR/***cas* **defense in LAB**

The CRISPR/*cas* defense system was first described in the 1980s for *E. coli,* but only recently recognized for lactic acid bacteria (2007), including such genera as *Lactobacillus*, *Bifidobacterium*, *Symbiobacterium, Enterococcus* and *Streptococcus*. Examination of more than 100 genomes of various LAB species allowed identifying over 60 different CRISPR loci, which were grouped into eight distinct families [155]. This indicates the highly diverse nature of LAB CRISPR loci. Additionally, it was observed that clustering of LAB CRISPRs was not in accordance with the classical phylogenetic correlations observed between the LAB phyla. This strongly implies that dissemination of CRISPR loci within the Prokaryotic world into separate lineages occurred by horizontal gene transfer events and their further evolution was imposed by the selective pressure due to phage infections. In general, CRISPR loci were determined to be located on the chromosome, except for one *E. faecium* strain found to carry the CRISPR array on a plasmid. Most LAB species harbor more than one CRISPR locus; yet, despite the common occurrence of CRISPR/*cas* systems, they have still not been identified for such species as *Lactococcus*, *Leuconostoc*, *Carnobacterium*, *Pediococcus*, and *Oenococcus*. This surprising absence of CRISPR loci was implied to be connected with an insufficient amount of sequencing data for these species in public databases. Examination of other strains of these species, involving genome sequencing, should be performed in order to fully resolve the issue on the existence of CRISPR/*cas* systems in these LABs. The identified various CRISPR arrays were determined to contain in total 100 different spacer sequences, including sequences of phage (26%) or prophage (47%) origin.

As CRISPR/*cas* systems confer phage resistance to host cells, they are quite of interest for the dairy industry where microbial production plays a significant role. Application of CRISPR/*cas* systems for construction of new LAB strain variants with differentiated resistance to phage infections is a novel alternative approach [67,142,156]. Moreover, such strains are regarded as safer for industrial applications, as the possibility for them to incorporate or disseminate foreign mobile genetic elements of unknown impact is low. Natural methods of selecting CRISPR-containing BIM cells (see section: 12.2.) of industrially applied bacteria could be an interesting solution for obtaining resistant strains, without deliberate genetic modifications. The first report on isolating CRISPR-containing lactic acid bacteria came from Barrangou et al. (2007) [67], who described the an approach of obtaining spontaneous *S. thermophilus* BIM cells by providing selection pressure due to phage infection. Protocols of isolating such strains have been later developed for dairy *S. thermophilus*, applied in the manufacturing of cheese and yoghurts [141]. The strategy is based on exposition of bacterial starter culture to high phage titers. Several rounds of growth in milk media under the constant selection pressure due to the phage presence resulted in obtaining phage-resistant mutants able to efficiently grow under industrial conditions. The great advantage of such approach is the fact that the presence of naturally acquired spacer sequences renders the strain resistant to phage infections, while preserving the industrially-attractive features of the initial starter cultures. Another strategy of constructing phage-resistant strains could be deliberate integration of synthetic spacers homologous to conserved sequences of industrial phage isolates into the CRISPR array of starter bacteria. However, this approach would involve certain molecular manipulations at the DNA level. Nonetheless, controlled modification of phage resistance of LAB strains using the CRISPR/*cas* regions is not considered by the food industry as a genetic modification method within the meaning of the existing rules in this area.
