A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496

Abstract

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

Keywords: Streptococcus thermophilus; bacteriophages; cryptic prophage; lipoprotein (Ltp); noncoding region.

Figure 1

Genome map of the TP1-M17PTZA496. The linear genome was circularized to improve its visualization. CDS, ORF, BLAST against the Streptococcus phage 20617, GC content, GC skew+, and GC skew−, are reported in circles from the outside inwards. Streptococcus phage 20617 whole genome sequence was used as a reference for the BLAST analysis. Only the ORFs between the attL and attR are displayed for the TP1-M17PTZA496.

Figure 2

Phylogenomic tree constructed using the whole genome sequence of the TP1-M17PTZA496, 83 S. thermophilus and 5 L. lactis bacteriophages. GenBank accession numbers are reported in Table S1. The scale bar represents a 1% difference on the average tBLASTx score.

Figure 3

Sequence alignment among the Streptococcus phage 20617, TP1-M17PTZA496, and the members of the genus Sfi11virus. Gray shading corresponds to the percentage of identity of the nucleotide sequences.

Figure 4

Phage-induction evaluation performed using four different chemicals: (A) Mitomycin C (1–4 µg/mL); (B) NaCl (100, 200, and 400 mM); (C) Nalidixic acid (0.1, 0.2, and 0.4 µg/mL); (D) H₂O₂ (100, 200, and 400 mM); (E) Lactose (0.1, 0.25, 0.5, and 1.0% w/v); (F) Sucrose (0.1, 0.25, 0.5, and 1.0% w/v). The same legend was used for different concentrations of the MmC (2–4 µg/mL) and the Nalidixic acid (0.1–0.2 µg/mL), since these growth curves overlapped each other.

Figure 5

(A) Semi-quantitative PCR analysis showing the MCP levels at 30, 60, and 90 min, after addition of different amounts of MmC. M17PTZA496_S71U was used as the bacterial chromosome control region. (B) attL and attR sites amplification to check the absence of phage excision. T0, T1, T2, and T3 are the sampling times, namely 0, 30, 60, and 90 min, respectively under diverse MmC concentrations (1–4 µg/mL).

Figure 6

(A) Phage titration of the S. thermophilus cultures. Samples were taken at three different time points, namely at 0 (T0), 30 (Pt1), and 60 min (Pt2). (A) After addition of the VA460 plus MmC. (B) S. thermophilus M17PTZA496 viable cell counts, after PBS (Cntr1), VA460 (Pt1), Mitomycin C (Cntr2 and Pt2), and VA460 plus MmC (Pt2) addition. Asterisks indicate different levels of statistical significance (*: p ≤ 0.05; ****: p < 0.0001).