Phage defence loci of Streptococcus thermophilus-tip of the anti-phage iceberg?

Abstract

Bacteria possess (bacterio)phage defence systems to ensure their survival. The thermophilic lactic acid bacterium, Streptococcus thermophilus, which is used in dairy fermentations, harbours multiple CRISPR-Cas and restriction and modification (R/M) systems to protect itself against phage attack, with limited reports on other types of phage-resistance. Here, we describe the systematic identification and functional analysis of the phage resistome of S. thermophilus using a collection of 27 strains as representatives of the species. In addition to CRISPR-Cas and R/M systems, we uncover nine distinct phage-resistance systems including homologues of Kiwa, Gabija, Dodola, defence-associated sirtuins and classical lactococcal/streptococcal abortive infection systems. The genes encoding several of these newly identified S. thermophilus antiphage systems are located in proximity to the genetic determinants of CRISPR-Cas systems thus constituting apparent Phage Defence Islands. Other phage-resistance systems whose encoding genes are not co-located with genes specifying CRISPR-Cas systems may represent anchors to identify additional Defence Islands harbouring, as yet, uncharacterised phage defence systems. We estimate that up to 2.5% of the genetic material of the analysed strains is dedicated to phage defence, highlighting that phage-host antagonism plays an important role in driving the evolution and shaping the composition of dairy streptococcal genomes.

Graphical Abstract

Figure 1.

(A) DNAPlotter view of chromosomes of S. thermophilus 4021 (outside track), 4145 (middle track) and 90730 (inside track) with CRISPR-Cas regions indicated in black, R/M systems (red), Abi systems (D or E; orange), PD-T4-6 (light blue, all), GAO_19 (yellow, 4021 and 4145), Kiwa (green, 90730) and Gabija (pink, 4145) highlighted in the relevant genomes where they occur. The CR-associated Defence Islands are highlighted in orange boxes with the annotated functions of flanking regions presented in blue boxes. (B) CR3-Defence Island harbours CR3, R/M system(s) and Gabija, Kiwa, GAO_19 and certain AbiD-encoding genes, where they occur.

Figure 2.

Upper panel: Identified R/M systems (both active systems and inactive systems/individual genes). Methylated bases within the detected motifs based on SMRT sequencing are indicated in blue text in the ‘motif’ column. Predicted m5C (pink text) and m6A (blue text) associated enzymes which were predicted but where methylation activity was not detected in this study. Where more than one gene was identified, the number of such genes is indicated by a number in the relevant box. In the ‘Enzyme’ column, black text indicates enzymes for which associated methylated motifs were identified, blue and pink text indicates enzymes for which m6A and m5C recognition sequences, respectively, were predicted but methylated motifs were not detected. Lower panel: Efficiency of plaquing (EOP) of lactococcal phages in the presence (induced) or absence (uninduced) of Type I, Type II or Type III R/M systems. In the uninduced state, there is no observable phage-resistance while in the induced state, all three evaluated R/M systems were observed to elicit substantial resistance against the evaluated phages 62606, 712, jj50, p2, c2 and sk1.

Figure 3.

(A) Schematic depiction of the gene(s) associated with the identified anti-phage systems and their co-location with R-M systems, where relevant. (B) Phage resistance level provided by the evaluated anti-phage systems tested against phages that infect S. thermophilus (Brussowvirus SW13; Moineauvirus STP1; P738 namesake and; 987 group phage SW16) and L. cremoris (Skunavirus sk1 and Ceduovirus c2). The log fold resistance is colour-coded according to the colour scale bar at the bottom left of the table. § indicates where the average plaque size reduction was observed in the presence of the anti-phage system relative to the wild type strain.

Figure 4.

Structure prediction of the SIR2-HerA proteins of the GAO_19 system. (A) Ribbon structure of 4021–0687 (rainbow colored). (B) Superposition of 4021–0687, SIR2₄₀₂₁ (rainbow colored) with PDB 1ici (grey), a NAD-dependent protein deacetylases (Z = 14.0, rmsd = 3.3Å). with the NAD residue (in sphere mode) from 1ici in the catalytic crevice. (C) Ribbon structure of the three domains 4021–0688, HerA₄₀₂₁ (rainbow colored). (D) Superposition of 4021-0688 (rainbow colored) with PDB 4d2i (grey), a HerA ATPase from Sulfolobus solfataricus (Z = 29.4, rmsd = 4.2 Å). (E) molecular surface representations (side and top views) of 4021–0688 hexamer compared to (F) the molecular surface of the HerA ATPase from Su. solfataricus. A–F): the ‘rainbow’ color coding consists of applying rainbow colors to the protein ribbon representation from blue (N-terminus) to red (C-terminus).

Figure 5.

Structure prediction of the AbiE proteins. (A) Ribbon structure of the two domains AbiEi (rainbow colored). (B) Superposition of AbiEi (rainbow colored) with PDB 6y8q (grey), an antitoxin from S. agalactiae (Z = 20.6, rmsd = 2.7 Å). (C) Ribbon structure of AbiEii (rainbow colored). (D) Superposition of AbiEii (rainbow colored) with PDB 6j7n (grey), a guanylyltransferase-like toxin from Mycobacterium tuberculosis (Z = 7.0, rmsd = 1.8 Å). The red dot line indicates the catalytic crevice and the red and blue dots the positions of the catalytic residues in AbiEii and 6j7n.

Figure 6.

(A) Distribution of anti-phage systems identified in S. thermophilus in RefSeq database based on DefenseFinder analysis. The number of occurrences of the system in the RefSeq database of 462 entries is shown on the Y-axis and the systems are indicated on the X-axis. The bars are colour-coded to indicate the proportion of genomes in the RefSeq database with the respective antiphage system in the species where purple = 90–100; dark orange = >20; light orange = 5–19 and; pale yellow = <5. (B) Schematic depicting the phage-resistance landscape of the analysed 27 S. thermophilus strains’ genomes. The presence of systems is indicated by a coloured box and absence of the systems is indicated by a white box. Each system is coloured differently. The genomes of these strains harbour an average of seven anti-phage systems based on this analysis.