A Decade of Streptococcus thermophilus Phage Evolution in an Irish Dairy Plant

Abstract

Phages of Streptococcus thermophilus present a major threat to the production of many fermented dairy products. To date, only a few studies have assessed the biodiversity of S. thermophilus phages in dairy fermentations. In order to develop strategies to limit phage predation in this important industrial environment, it is imperative that such studies are undertaken and that phage-host interactions of this species are better defined. The present study investigated the biodiversity and evolution of phages within an Irish dairy fermentation facility over an 11-year period. This resulted in the isolation of 17 genetically distinct phages, all of which belong to the so-called cos group. The evolution of phages within the factory appears to be influenced by phages from other dairy plants introduced into the factory for whey protein powder production. Modular exchange, primarily within the regions encoding lysogeny and replication functions, was the major observation among the phages isolated between 2006 and 2016. Furthermore, the genotype of the first isolate in 2006 was observed continuously across the following decade, highlighting the ability of these phages to prevail in the factory setting for extended periods of time. The proteins responsible for host recognition were analyzed, and carbohydrate-binding domains (CBDs) were identified in the distal tail (Dit), the baseplate proteins, and the Tail-associated lysin (Tal) variable regions (VR1 and VR2) of many isolates. This supports the notion that S. thermophilus phages recognize a carbohydrate receptor on the cell surface of their host.IMPORTANCE Dairy fermentations are consistently threatened by the presence of bacterial viruses (bacteriophages or phages), which may lead to a reduction in acidification rates or even complete loss of the fermentate. These phages may persist in factories for long periods of time. The objective of the current study was to monitor the progression of phages infecting the dairy bacterium Streptococcus thermophilus over a period of 11 years in an Irish dairy plant so as to understand how these phages evolve. A focused analysis of the genomic region that encodes host recognition functions highlighted that the associated proteins harbor a variety of carbohydrate-binding domains, which corroborates the notion that phages of S. thermophilus recognize carbohydrate receptors at the initial stages of the phage cycle.

Keywords: Streptococcus; bacteriophage; dairy industry; genomics; receptor binding protein.

FIG 1

Unrooted phylogenetic tree of the 17 sequenced isolates and a representative cos (DT1), pac (TP-J34), 5093 (5093), and 987 (9871) groups included as references. All phage isolates group most closely to DT1, while distinct subgroups or lineages (L) of the phage isolates are also highlighted in the tree (L1 to L4). Factory-derived phage isolates are indicated in black text, externally derived phage isolates are indicated in green text, and representatives of the four S. thermophilus groups (cos, pac, 5093, and 987) are indicated in blue text.

FIG 2

Schematic representation of the genomes of lineage 1 (STP1, STP2, C0, A0, 7A5, B5, B0, and L5A1) and lineage 2 (7T, 9B4, 31B4, 16B8, R1, and V2) phages. Arrows (indicating protein-encoding regions) joined by shaded boxes indicate genetic regions of similarity, with the black shading indicating 90 to 100%, dark gray indicating 80 to 89%, light gray indicating 50 to 79%, and off-white indicating 30 to 49% aa identity. Arrows of the same color represent genes with a similar function. Gray arrows are indicative of genes encoding proteins of unknown function. The predicted functions of the encoded proteins are presented above the relevant arrows, where known and the functional modules are presented below the schematic. The major region of divergence between 7T (the first lineage 2 isolate) and STP1 (first lineage 1 isolate) is highlighted in the 7T genome representation by a red box. Similarly, regions of genetic novelty associated with isolates B5, 7A5, and L5A1 are highlighted in red boxes. All lineage 2 phage genomes possess a genomic region with greater than 90% identity. The lineage (L1/2) and source (F, factory; E, external) are also presented on the left side of the figure. LTR, long terminal repeat.

FIG 3

Schematic representation of the genomes of lineage 3 (MM25 and 9A) and lineage 4 (M19) phages and their comparison to the first isolate of the study (STP1, lineage 1). Arrows (indicating protein-encoding regions) joined by shaded boxes indicate genetic regions of similarity, with the black shading indicating 90 to 100%, dark gray indicating 80 to 89%, light gray indicating 50 to 79%, and off-white indicating 30 to 49% aa identity. Arrows of the same color represent genes with a similar function. Gray arrows are indicative of genes encoding proteins of unknown function. The predicted functions of the encoded proteins are presented above the relevant arrows, where known and the functional modules are presented below the schematic. The lineage (L3/4) and source (F, factory; E, external) are also presented on the left side of the figure.

FIG 4

Top, unrooted phylogenetic tree of the Tal-RBPs of the 17 sequenced phages highlighting the disparity of the Tal-RBPs of the lineage 1/2 (left) and 3/4 phages (right). Bottom, schematic depicting the organization of the Tal-RBPs of the sequenced phages using representatives of the group. All Tal-RBPs possess a conserved N-terminal ∼400-aa Tal domain (blue). Phage 9A (lineage 3) is the sole phage encoding a Tal-RBP with two BppA-like (5E7T_B) CBDs (purple), and these constitute the regions described as VR1 and VR2. VR1, where present, is always flanked by collagen repeat motifs (red). The VR2 region may/may not incorporate a 5E7T_B BppA CBD (purple) and/or may represent a distinct CBD, e.g., Igu1_A domain (yellow), or a CBD of unidentified structure. STP1 is representative of the Tal-RBPs of lineage 1 and 2 phages, which all share a similar size and architecture. MM25 is representative of the lineage 4 phages.

FIG 5

Representative electron micrographs of STP1 (A) and MM25 (B). Both phages display long tails with protruding feather-like appendages from the tail tip.