Distribution of clustered regularly interspaced palindrome repeats CRISPR2 and CRISPR3 in Lactobacillus delbrueckii ssp. bulgaricus strains

The function of several families of clustered regularly interspaced palindrome repeats (CRISPRs) in prokaryotic genomes was recently found to be related to the protection of bacterial cells against the expression of foreign DNA, originating from plasmids or bacteriophages. The present study was the first attempt to screen a broader number of Lactobacillus delbrueckii ssp. bulgaricus strains, widely used in yoghurt and cheese production, for the presence of CRISPRs. Database search of four completely sequenced L. delbrueckii ssp. bulgaricus genomes indicated the presence of CRISPR2 in three of them - ATCC 11842, ATCC BAA-365 and ND02, and the presence of CRISPR3 in strain 2038. In the first three strains, the CRISPR2 was invariably located between a 3′–5′ exonuclease gene and a gene for a ppGpp-synthetase. The location of CRISPR3 in strain 2038 was between a histidine-kinase gene and an acetyl-CoA acetyltransferase gene, 2 kbp downstream of the CRISPR2 locus in ATCC 11842. Specific primers were designed to amplify with polymerase chain reaction the target regions containing the potential CRISPR2 and / or CRISPR3 in a total of 33 L. delbrueckii ssp. bulgaricus strains. Thirteen strains yielded a high molecular mass product corresponding in size and location to CRISPR2 of the type strain ATCC 11842, while another 17 strains indicated the presence of potential CRISPR3, analogous to that of strain 2038. Three strains did not indicate the presence of CRISPRs. Interestingly, none of the tested strains carried both CRISPR2 and CRISPR3 simultaneously in its genome at the investigated region.