**Author details**

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

bacterial populations.

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

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

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 A.K. Szczepankowska, R.K. Górecki and J.K. Bardowski *Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Warsaw, Poland* 

P. Kołakowski \* *Danisco Biolacta, Innovation, Olsztyn, Poland* 
