Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization

Abstract

Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.

Keywords: CRISPR; CRISPR-Cas; Streptococcus thermophilus; bacteriophage morphogenesis; bacteriophages; electron microscopy; evolution; genomic analysis; lactic acid bacteria; tail protein.

FIG 1

TEM micrographs of CsCl-purified P738 (left) and D4446 (right) phage particles stained with uranyl acetate (bar indicates 100 nm) with general characteristics for each phage. The arrows indicate the distal ends of the thin (twisted) tail fibers.

FIG 2

Pairwise BLASTn comparison of the two new S. thermophilus phages P738 and D4446 and S. pyogenes prophage T12. Analysis shows high relatedness between phages P738 and D4446 and homology with prophage T12. Genes identified by bioinformatics analysis as belonging to specific modules are color-coded. Selected genes are annotated in the figure, encoding conserved proteins. TerS, terminase small subunit; TerL, terminase large subunit; TMP, tail tape measure protein; Dit, distal tail protein; Tal, tail-associated lysin.

FIG 3

Schematic illustration representing the alignment of the S. thermophilus phage P738 genome with representative ORFs of other S. thermophilus phages. VR1, VR2, and VR3 indicate variable regions 1, 2, and 3, respectively. TerS, terminase small subunit; TerL, terminase large subunit; TMP, tail tape measure protein; Dit, distal tail protein; Tal, tail-associated lysin.

FIG 4

Full-sequence HHpred analysis of the insertions of evolved Dit (A) and Tal (B) proteins of phages D4446 and P738. The HHpred output in red means that the HHpred probability is over 98%. The label indicates which Protein Data Bank (PDB) entry is closest to the query.

FIG 5

Schematic depiction of the identical architecture of the distal tail and Tal regions of phages D4446 and P738. (based on HHpred analysis). The Dit structural ring and galectin domains (top) are colored green. The Dit CBMs 1 and 2 are colored yellow and purple, respectively. The Tal structural parts are colored brown/beige. The Tal N-terminal CBM insertions 1 and 2 are colored orange and red, respectively, and those on the Tal extension (CBMs 3 and 4) are colored blue and beige, respectively.

FIG 6

Proteome-based phylogenetic tree using the Genome-BLAST Distance Phylogeny (GBDP) method with VICTOR under settings recommended for prokaryotic viruses. S. thermophilus phages are colored according to their genus, and S. pyogenes phage T12 was included to root the tree.

FIG 7

CRISPR array comparison. (A) A set of 35 S. thermophilus strains are displayed in clusters of similar arrays. CRISPR 1 and CRISPR 3 are shown from right to left. The color-coding distinguishes the different groups after clustering, with the array clustering method implemented in CRISPRStudio. (B) Unique spacers present only once across the data set are colored, and spacers present at least twice are grayed out to show the S4 array’s originality.