Comparative Genomics of Closely Related Tetragenococcus halophilus Strains Elucidate the Diversity and Microevolution of CRISPR Elements

Abstract

Tetragenococcus halophilus – a halophilic lactic acid bacterium – is frequently used as a starter culture for manufacturing fermented foods. Tetragenococcus is sometimes infected with bacteriophages during fermentation for soy sauce production; however, bacteriophage infection in starter bacteria is one of the major causes of fermentation failure. Here, we obtained whole-genome sequences of the four T. halophilus strains YA5, YA163, YG2, and WJ7 and compared them with 18 previously reported genomes. We elucidated five types of clustered regularly interspaced short palindromic repeat (CRISPR) loci in seven genomes using comparative genomics with a particular focus on CRISPR elements. CRISPR1 was conserved in the four closely related strains 11, YA5, YA163, and YG2, and the spacer sequences were partially retained in each strain, suggesting that partial deletions and accumulation of spacer sequences had occurred independently after divergence of each strain. The host range for typical bacteriophages is narrow and strain-specific thus these accumulation/deletion events may be responsible for differences in resistance to bacteriophages between bacterial strains. Three CRISPR elements, CRISPR1 in strains 11, YA5, YA163, and YG2, CRISPR2 in strain WJ7, and CRISPR2 in strain MJ4, were inserted in almost the same genomic regions, indicating that several independent insertions had occurred in this region. As these elements belong to class 1 type I-C CRISPR group, the results suggested that this site is a hotspot for class 1, type I-C CRISPR loci insertion. Thus, T. halophilus genomes may have acquired strain-specific bacteriophage-resistance through repeated insertion of CRISPR loci and accumulation/deletion events of their spacer sequences.

Ribitol-Containing Wall Teichoic Acid of Tetragenococcus halophilus Is Targeted by Bacteriophage phiWJ7 as a Binding Receptor

“…In conclusion, it was estimated that WJ7 is endowed with gene sets for both Gro-WTA and Rbo-WTA synthesis. We compared the tagAB -containing locus with three other strains, YA5, YA163, and YG2, for which draft genome sequences are available ( 5 ). The three strains did not contain tarIJL in their genomes, similar to NBRC 12172, and all of them were insusceptible to phiWJ7 (Fig.…”

“…The previously published genome sequence of the wild-type strain T. halophilus WJ7 (GenBank accession numbers BLRM01000001 to BLRM01000113 ) was used as the reference sequence for our genome mapping analysis ( 5 ). The Illumina sequence reads of these strains were mapped to the reference sequence using BWA with default parameters ( 38 ).…”

“…Higuchi et al successfully generated phage-insensitive mutants from T. halophilus D10, but the mechanisms that altered phage susceptibility were not revealed ( 4 ). Recently, CRISPR sequences were found in the genomes of T. halophilus and were proposed as one of the defense systems against bacteriophages ( 5 ). However, the factors that determine the phage susceptibility of this species were not experimentally demonstrated.…”

Identification of Capsular Polysaccharide Synthesis Loci Determining Bacteriophage Susceptibility in Tetragenococcus halophilus

“…To detect the mutation sites of YA5R1, YA5R1R1, and YG2R1, the genome data for T. halophilus YA5, YA5R1, and YG2 were used as the reference sequences for our genome mapping analysis. The genome sequences of YA5 (DRA accession number DRR424329 ) and YG2 (accession number DRR220997 ) were previously published ( 56 ). The Illumina sequence reads of the derivatives were mapped to the reference sequence of the parental strain using BWA with default parameters ( 57 ).…”