Evolution of CRISPR1 and CRISPR3 in Spontaneous Phage-Resistant Mutants of Streptococcus Thermophilus Strain LBB.A

Abstract

ABSTRACTStreptococcus thermophilus LBB.A is a component of industrial starters for traditional Bulgarian yogurt. Eight genetically distinguishable cos-type phage cultures: φ(variant);A1, φ(variant);A2, φ(variant)A5, φ(variant);A6, φ(variant);DmA, φ(variant);SfA, φ(variant);PtA, and φ(variant);A7, were found to infect strain LBB.A. Bacteriophage-Resistant Mutants (BIMs) of strain LBB.A were isolated and their Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR1 and CRISPR3) were amplified by PCR and compared to the parent strain.Challenging S. thermophilus LBB.A with φ(variant);A1 resulted in the isolation of “first order” BIM AR3A1, which was resistant to six of the phages, but remained sensitive to φ(variant);PtA and φ(variant);A7. In AR3A1 the size of the CRISPR1 and CRISPR3 loci increased with approximately 66 bp each, compared to the parent strain, corresponding to the insertion of one additional repeat unit. Next, AR3A1 was challenged with φ(variant);A7 to obtain “second order” BIM AR3-3A1A7, which was resistant to all of the eight phages. Strain AR3-3A1A7 was found to have acquired only another 65 bp in its CRISPR1 locus with no change in CRISPR3. The newly acquired sequences in CRISPR1 and CRISPR3 of AR3A1 and AR3-3A1A7 showed similarity to sequences in already published S. thermophilus phage genomes. The phage-resistant mutant AR3-3A1A7 acidified milk at a rate comparable to the parent strain LBB.A and, consequently, AR3A1A7 was recommended as a potential replacement of the sensitive parent strain in yogurt production. The difference in the size of the amplified region of CRISPR1 and CRISPR3 can be used as a genetic marker to differentiate between BIMs and their phage sensitive parent strain.