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

Philip Kelleher, Guillermo Ortiz Charneco, Zoe Kampff, Natalia

Diaz-Garrido, Francesca Bottacini, Brian Mcdonnell, Gabriele A Lugli, Marco

Ventura, Alexey Fomenkov, Pascal Quénée, et al.

► To cite this version:

Philip Kelleher, Guillermo Ortiz Charneco, Zoe Kampff, Natalia Diaz-Garrido, Francesca Bottacini, et al.. Phage defence loci of Streptococcus thermophilus- tip of the anti-phage iceberg?. Nucleic Acids Research, 2024, 52 (19), pp.11853-11869. 10.1093/nar/gkae814. hal-04910547

HAL Id: hal-04910547

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

2 **Running title:** Phage resistance systems of *Streptococcus thermophilus*

3 Philip Kelleher¹, Guillermo Ortiz Charneco¹, Zoe Kampff¹, Natalia Diaz-Garrido¹, Francesca

4 Bottacini², Brian McDonnell¹, Gabriele A. Lugli³, Marco Ventura³, Alexey Fomenkov⁴, Pascal

5 Quénée⁵, Saulius Kulakauskas⁵, Paul de Waal⁶, Noël N.M.E. van Peij⁶, Christian Cambillau¹,⁷,

6 Richard John Roberts⁴, Douwe van Sinderen¹*, Jennifer Mahony¹*

7

8 ¹School of Microbiology and APC Microbiome Ireland, University College Cork, Cork, T12

9 YT20 Ireland (ORCID P.K. 0000-0003-3423-7930; G.O.C. 0000-0003-1601-6173; Z.K. 0000-0002-8399-1465; N.D. 0000-0003-1220-015X; B.MD. 0000-0003-1704-8759; D.vS. 0000-0003-1823-7957; J.M. 0000-0001-5846-6303).

12 ²Department of Biological Sciences, Munster Technological University, Cork, Ireland (ORCID
13 F.B. 0000-0002-0142-2956).

14 ³Laboratory of Probiogenomics, Department of Chemistry, Life Sciences and Environmental Sustainability, and Interdepartmental Research Centre Microbiome Research Hub, University of Parma, Parma, Italy (ORCID G.A.L 0000-0002-3024-0537.; M.V. 0000-0002-4875-4560)

17 ⁴New England Biolabs, Ipswich, Massachusetts, United States of America (ORCID A.F. 0000-0002-2463-5946; R.J.R. 0000-0002-4348-0169)

19 ⁵Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France
(ORCID S.K. 0000-0002-3466-8266)

21 ⁶DSM-Firmenich, Taste, Texture & Health, Center for Food Innovation, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands (ORCID P.dW. 0000-0002-3521-7483; N.N.M.E.vP. 0009-0002-3946-4067)

24 ⁷Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IMM), Aix-Marseille Université – CNRS, UMR 7255, Marseille, France. (ORCID C.C. 0000-0001-5502-4729).

27 * To whom correspondence should be addressed: Jennifer Mahony & Douwe van Sinderen, School of Microbiology & APC Microbiome Ireland, University College Cork, Western Road, Cork, Ireland

30 Tel: +353 21 4902730/1365; Fax: +353 21 4903101; Email: j.mahony@ucc.ie ;

31 d.vansinderen@ucc.ie

32 **Keywords:** Dairy, starter culture, phage resistance, restriction and modification, methylation

33
34 **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.

52

53 **INTRODUCTION**

*Streptococcus thermophilus*, a member of the viridans group of streptococci, is widely exploited in the dairy industry owing to its associated technological properties (1). The intensive application of strains of this species and continuous cultivation under optimal growth conditions in contained industrial environments provides the ideal opportunity for (bacterio)phages to proliferate (2). Phages represent one of the most significant challenges to dairy fermentations as they cause production disruptions and may persist in the processing environment for extended time periods (3). Dairy streptococcal phages are classified into one of five genetically distinct groups: the *Moineauvirus* (formerly termed the *cos* group), *Brussowvirus* (formerly termed the *pac* group), *Vansinderenvirus* (formerly termed the 5093 group), 987 and P738 phages (4). In response to the persistent threat of phages, *S. thermophilus* strains have developed a collection of protective antiphage systems. It is widely
understood that the defence systems of dairy streptococci predominantly include clustered
regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes
as well as restriction and modification (R/M) systems (5, 6). Furthermore, it has been shown
that these systems work compatibly to increase the overall phage-resistance level of the host
strain (7).

Beyond CRISPR-Cas and R/M systems, there are limited reports of other phage-
resistance systems being active in *S. thermophilus*. These include the prophage-encoded
lipoprotein Ltp system that was first described in *S. thermophilus* (pro)phage TPJ-34 (8). This
system interferes with the process of phage DNA injection and was shown to provide
resistance against both dairy streptococcal and lactococcal phages. Homologues of *ltp* have
subsequently been identified in other *S. thermophilus* prophages and while the incidence of
prophage carriage among strains of this species is reportedly low, it highlights the potential
benefits conferred by integrated temperate phages (5, 9). The phenomenon of phage-
resistance systems providing protection in both streptococci and lactococci is not limited to
this system. For example, the lactococcal abortive infection (Abi) system AbiA provides
resistance against virulent phage infection in *S. thermophilus* strains at 30 °C. Interestingly,
this protective effect was abolished at temperatures at and above 37 °C (10). Although the
latter corresponds to the optimal growth temperatures for *S. thermophilus* in food
fermentations, strains of this species will grow at 30 °C and when applied as an adjunct in
mesophilic fermentations, the activity of such cross-species functionality may present a
considerable benefit to the robustness of the process.

In recent years, a wide range of novel phage-resistance systems have been identified in both
Gram-positive and Gram-negative bacteria (11–15). The identification of so-called "Defence
Islands" on bacterial chromosomes has highlighted the presence of genomic regions
dedicated to providing protection against phages (11, 16, 17). In *E. coli* many such defence
hotspots were observed to be associated with mobile genetic elements and prophages (18).
In parallel, the availability of bioinformatic tools to detect the presence of phage-resistance
systems in bacterial genomes has significantly improved, in particular through tools such as
PADLOC, MacSyFinder and Defensefinder (19–23). While *S. thermophilus* is a relatively young
species (estimated 3,000 to 30,000 years), its adaptation to growth in milk has resulted in
extensive genome decay and horizontal gene transfer events has facilitated its diversification
(24). Given the significant pressure imposed on strains of this species by phages, it is very likely that the consistent presence in fermentation facilities has selected for isolates that acquired genomic regions that encode phage-resistance systems. In the present study, the complete genomes of 27 S. thermophilus strains were sequenced using a Pacific Biosciences (PacBio) platform to evaluate the resistome landscape including functional analysis of a selection of predicted phage resistance systems and their functionality in both Lactococcus cremoris and S. thermophilus.
120 MATERIALS AND METHODS

121 Biological resources and culturing conditions

122 All *S. thermophilus* strains whose genomes were sequenced as part of this study are listed in
123 Table 1. The strains that were selected for genome sequencing in this study are all dairy
124 isolates and have been applied and/or characterized in previous studies (25–28).

125 Furthermore, in a recent survey of the genetic diversity of the gene cluster associated with
126 rhamnose-glucose polysaccharide (RGP) biosynthesis in *S. thermophilus*, the selected strains
127 were shown to represent diverse genotypic groups (25). *S. thermophilus* Moz109 was
128 furthermore used as a host for the evaluation of phage-resistance activity. *S. thermophilus*
129 strains were grown in M17 broth (Oxoid Ltd., U.K.) supplemented with 0.5 % lactose and
130 incubated without agitation at 42 °C for 16-18 hours. *Lactococcus cremoris* NZ9000 was
131 grown in M17 broth supplemented with 0.5 % glucose and incubated at 30 °C for 16-18
132 hours without agitation.

133 Isolation of genomic DNA, sequencing, assembly and annotation

134 Genomic DNA was isolated from 27 *S. thermophilus* strains using the Macherey-Nagel
135 NucleoBond system, utilising Buffer Set III and AXG or AX 100 columns (Macherey-Nagel,
136 Germany). This was performed according to the manufacturer's instructions with the
137 following modifications: at the cell lysis step, the volume of lysozyme (100 mg/mL) added
138 was increased to 40 µL, and 50 µL mutanolysin (5,000 units/mL; Sigma-Aldrich, Germany) was
139 also added prior to incubation, which proceeded for an increased period of 1 hour. In
140 addition, where necessary, insoluble components were removed from the supernatant by
141 centrifugation up to 10,000 × g for 10 minutes prior to loading on the column. DNA quantity
142 was measured by Qubit 2.0 (Life Technologies, USA) and quality assessed by visual
143 inspection using agarose gel electrophoresis.

144 Genomic DNA was sheared to an average size of ~10 kb using the G-tube protocol (Covaris,
145 MA). DNA libraries were prepared using a SMRTbell express template prep kit 2.0 (100-938-
146 900, Pacific Bioscience, CA, USA) and ligated with hairpin barcoded lbc adapters.

147 Incompletely formed SMRTbell templates were removed by digestion with a combination of
148 exonuclease III and exonuclease VII (NEB). The qualification and quantification of the
149 SMRTbell libraries were made on a Qubit fluorimeter (Invitrogen, USA) and a 2100
150 Bioanalyzer (Agilent Technologies, USA). SMRT sequencing was performed using an SQ1
151 (Pacific Biosciences) instrument based on the multiplex protocol for 10 kb SMRTbell library
152 inserts or Pacific Biosciences SMRT RSII technology. Raw sequencing reads were assembled
153 using the hierarchical genome assembly process (HGAP) protocol RS_Assembly.2 in the
154 SMRT analysis software v2.3 using the default settings for RSII sequence data. Sequencing
155 reads emanating from the SQ1 sequencing instrument were de novo assembled using the
156 Microbial Assembly version 10.1.0.1119588 (29) program with default quality and read length
157 parameters. In addition to genome assembly, the SMRT Analysis pipeline from Pacific
158 Biosciences (http://www.pacbiodevnet.com/SMRT-Analysis/Software/SMRT-Pipe) enables the
159 determination of the epigenetic status of sequenced DNA by identifying m6A and m4C
160 modified motifs (30-32).

161 Genome annotation was performed by NCBI using the PGAP annotation pipeline (33).
162 Artemis (34) (v18) genome browser and annotation tool was used to inspect and, where
163 necessary, to manually curate predicted ORFs. ORF annotations were refined where
164 necessary using alternative databases including Interpro (35), HHpred (36) and Uniprot/EMBL
165 (37). All sequence comparisons at a protein level were performed via all-against-all, bi-
166 directional BLAST alignments. An alignment cut-off threshold of E-value <0.0001, with >30 %
167 amino acid identity across 80 % of the sequence length was applied.

168 Genome scan for potential phage-resistance systems and prophage regions

169 Primary scanning of the assembled genome for putative methyltransferase (MTase) genes
170 was performed using the Seqware program (38). Additional searches for MTase genes were
171 performed using HMMer (HMMER 3.3.2; http://hmmer.org/) to annotate each predicted
172 protein coding sequence (CDS) feature in the genomes. Genes matching MTase sequence
173 profiles were further examined by structure prediction using the ColabFold implementation
174 of AlphaFold2 (39, 40), followed by structure similarity search using predicted MTase models
175 as a query inputs to DALI (41).

176 CRISPR-Cas encoding regions were identified using CRISPRCasFinder
177 (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) and selecting the evidence level
178 2 to 4 outputs only (42). A general search for phage-resistance systems (including and
beyond R/M and CRISPR-Cas systems) in the genomes of the 27 strains sequenced as part of this study was performed using PADLOC (https://padloc.otago.ac.nz/padloc/) using default settings (19). Where more than one strain was identified to encode similarly predicted phage-resistance systems (e.g. AbiD, AbiEi/AbiEii and Sirtuin-dependent system), the sequences of the similarly annotated systems were compared to each other using BLASTp analysis. Where a single example of a predicted phage-resistance system was identified (e.g. Hachiman, Gabija and Kiwa), a BLASTn search was performed against the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To establish the distribution of phage-resistance systems in *S. thermophilus* strains' genomes that are available in the RefSeq database, DefenseFinder was used with a search term of "Streptococcus thermophilus" and visualizing the heat map under the species level of taxonomic rank (22). The distribution of systems was reported as the number of systems identified in the RefSeq database (from a pool of 462 RefSeq entries) while the heatmap reported the proportion of genomes presenting with a given system per species taxonomic rank.

Prophage regions were predicted using PHASTEST using default settings (43). Prophage regions are predicted by PHASTEST as intact, incomplete or questionable prophages. The identified regions were manually inspected to establish the gene content of the predicted prophages.

CR-Defence Islands were defined using the following criteria: (1) the CRISPR loci were used as the "anchor" of possible defence islands and (2) the surrounding gene content was considered part of the defence island until conserved genes/gene clusters were reached and which were considered the outer boundaries of the proposed defence island. In this manner, three defence island regions were identified, CR1-Defence Island, CR2/4 Defence island and CR3-Defence Island.

## Construction of an R/M negative derivative of *L. cremoris* NZ9000

An *L. cremoris* NZ9000 mutant carrying deletion of a 5,945 bp DNA fragment encoding a complete Type I R/M system (comprising *llnz_03405*, *llnz_03410*, and *llnz_03415* which encode HsdR, M, and S, respectively) was constructed by double-crossover (DCO) recombination, using the plasmid pGhost9-ΔhsdRMS containing the *hsdRMS* flanking DNA fragments. To construct this plasmid, two PCR products of approximately 700 bp of the
209 flanking regions of *hsdRMS* were amplified from NZ9000 using two pairs of primers
210 (034RM1-034RM2, and 034RM3-034RM4, described in Supplementary Table S1) and Phusion
211 high fidelity DNA polymerase (New England Biolabs). After purification, the PCR products
212 were fused to thermosensitive plasmid pGhost9, digested with SmaI (New England Biolabs),
213 by the strand overlap extension method, using the Gibson assembly cloning kit (New
214 England Biolabs). The reaction mixture was introduced into *E. coli* JIM4646 with appropriate
215 selection (Ery). For one such clone (which we named VES7817), the DNA sequence of the
216 cloned fragment of pGhost9-ΔhsdRMS was verified by PCR and sequencing, using primers
217 pGH9L and pGH9R (Supplementary Table S1). pGhost9-ΔhsdRMS was introduced to NZ9000
218 and presumptive transformant colonies were selected at the permissive temperature (30 °C)
219 on GM17 plates containing erythromycin (3 µg/ml; GM17+Ery3). To select single cross-over
220 events, a VES7817 overnight liquid culture (first grown in GM17+Ery3 at 30 °C) was plated on
221 GM17+Ery3 and incubated at the non-permissive temperature of 42 °C. The second
222 recombination event leading to plasmid excision was selected by inoculating resultant
223 colonies in liquid GM17 medium without antibiotic at 42 °C, and shifting exponentially
224 growing culture to 30 °C for 2 hours, followed by overnight growth at 42 °C. The culture was
225 maintained on GM17 agar plates without antibiotic selection at 30 °C. A strain harbouring a
226 *hsdRMS* deletion (termed VES7862) was selected as an erythromycin-sensitive clone and the
227 presence of the deletion in its chromosome was confirmed by PCR and sequencing using
228 primers 034RmdelL and 034RmdelR (Supplementary Table S1).

229 **Cloning and confirmation of R/M activity**

230 Cloning of selected R/M systems into the inducible vector pPTPi (44) was performed using
231 conventional recombinant DNA techniques (oligonucleotide primers are listed in
232 supplementary Table S1). Induction of this promoter was achieved by the addition of nisin (5
233 ng/ml) in the growth medium. Obtained pPTPi constructs were subsequently transformed
234 into the previously described R/M-free derivative *L. cremoris* VES7862. Confirmation of R/M
235 activity of these pPTPi constructs was conducted by inducing each construct, and the empty
236 vector control, with 5 ng/ml nisin and testing each strain for phage-resistance activity against
237 the lactococcal *Skunavirus* sk1 and *Ceduovirus* c2 using an established double agar plaque
238 assay method (45).
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## Evaluation of phage-resistance activity of non-R/M, non-CRISPR systems

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Representative genes that encode homologues of AbiD (St55_0646, St4078_0760, St4021_1632), AbiEi/AbiEii (St90730_1386, St90730_1387), Hachiman (St19_0447, St19_0448), Kiwa (St90730_688, St90730_689), Sirtuin-dependent system (St19_0691, St19_0692) and Gabija (St4145_0685, St4145_0686) were cloned into the high copy number plasmid pNZ44 under the control of the constitutive lactococcal P44 promoter (46). For systems incorporating predicted gene pairs, both genes were cloned in tandem. The primer pairs used for the amplification of the target genes are presented in Supplementary Table S1. The sequence of the generated constructs was verified by Sanger sequencing (Genewiz, Leipzig, Germany). The constructs were introduced into L. cremoris NZ9000, S. thermophilus 4078 and S. thermophilus Moz109 by electroporation. The empty vector was also introduced into the three strains as a control for phage assays. Phage-resistance activity elicited by the predicted phage-resistance systems was evaluated using plaque assays (45) and the efficiency of plaquing (E.O.P.) of phages was calculated by dividing the phage titre on the test strain (i.e. strains carrying the predicted phage-resistance system constructs) by the phage titre of the strain harbouring the empty vector. The Skunavirus sk1 and Ceduovirus c2 were used in plaque assays with the L. cremoris host strain NZ9000. The streptococcal Brussowvirus SW13 was used in plaque assays with S. thermophilus 4078 and the Moineauvirus STP1 was used in plaque assays with S. thermophilus Moz109. All assays were performed in (at least) triplicate and the presented results are the average of these data.

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## Alphafold analysis of phage-resistance systems

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Structure predictions of the identified putative phage-resistance protein(s) were performed with a Colab notebook running AlphaFold v2.3.1 (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb) or HPC resources from GENCI-IDRIS running AlphaFold v2.3.1 (40). The pLDDT values and the predicted alignment errors (PAE) were obtained from the Colab Notebook or the IDRIS calculations output. The pLDDT values were also stored in the B-factor column of the PDB files. The pLDDT and PAE plots for each protein of complex are provided as supplementary Figures S1 and S2. The final predicted protein or domain structures were submitted into the Dali server (41) to identify the closest structural homologs in the PDB. Dali
provides a root mean square deviation value (r.m.s.d) in Å, as well as an aggregated factor called Z-value. A Z-score above 20 means the two structures are definitely homologous, between 8 and 20 means the two are probably homologous, between 2 and 8 is a grey area, and a Z-Score below 2 is not significant. Visual representations of the structures were prepared with ChimeraX (47). The "rainbow" color coding consist of applying rainbow colors to the protein ribbon representation from blue, at the N-terminus, to red at the C-terminus. Proteins superpositions were performed using Dali superpositions (41).

276
278 **S. thermophilus** dedicates a significant portion of its genome to phage defence

279 The genomes of 27 *S. thermophilus* strains were sequenced to completion. The genomes
280 were shown to possess a G+C content of approximately 39 %, are between 1.78 and 1.94
281 Mbp in length and are predicted to harbour 1,821 to 2,023 coding sequences (Table 1).

282 These *S. thermophilus* genomes were analysed for the presence of phage-resistance systems
283 using PADLOC. The combined length of the genetic regions that are dedicated to the
284 predicted phage-resistance systems (excluding phage defence candidate systems) was
285 calculated and ranged between 17 and almost 45 kb representing 0.9-2.4 % of total bacterial
286 genetic content. These genomic regions primarily comprise of genes encoding CRISPR-Cas
287 (0.2-1.3 % of total genome) and R/M (0.1-0.7 % of total genome) systems; however,
288 homologues of lactococcal abortive infection (Abi) and more recently identified phage
289 defence systems from other bacteria were also identified in the analysed genomes with
290 further details presented below. Based on the current analysis, strains of *S. thermophilus* are
291 predicted to harbour an approximate average of seven phage-resistance systems per
292 genome with CRISPR-Cas and R/M systems representing their core phage resistome and
293 incorporating additional and, in many cases, strain-specific repertoires of phage-resistance
294 systems.

295 In addition to the chromosomally-located phage defence systems, we evaluated the
296 contribution of mobile genetic elements to the anti-phage repertoire in this species using
297 PADLOC. *S. thermophilus* strains do not typically harbour many, if any, plasmids, likely owing
298 to the presence of CRISPR-Cas systems in strains of this species. Among the strains analysed
299 in this study, plasmids were identified associated with strains ST1A, 4021, 4067, 4147,
300 CNRZ1202 (2 plasmids), AVA116, ST55 and ST128 (Table S2). These plasmids range in size
301 from ~3.4 – 8.2 kb and are predicted to harbour between two and seven ORFs (Table S2). Of
302 the nine identified plasmids, only one possessed an identifiable (complete) anti-phage
303 system, i.e. a Type II R/M system on pCNRZ1202A. Additionally, the genomes were analysed
304 for the presence of prophage-associated regions and their possible contribution to the
305 phage-resistance landscape. Using the outputs of Phastest, 12 of the 27 assessed *S.
306 thermophilus* genomes were predicted to have at least one prophage region, with one
307 genome (i.e. that of strain CNRZ385) possessing two predicted "questionable" prophage
regions (Table 2). Among the identified prophage regions, only one possesses an identifiable superinfection exclusion system, which may contribute to phage-resistance in the host strain (STR1). Nonetheless, all identified prophage regions harbour several transposase-encoding genes, highlighting the importance of these regions as possible recombination sites and repository for novel genetic acquisitions. Therefore, it appears that mobile genetic elements including prophages and plasmids are not a major source of anti-phage systems while chromosomal variations and acquisitions are dominantly associated with the defences of this species.

316

## The CRISPR-Cas landscape of *S. thermophilus*

Strains of *S. thermophilus* are reported to harbour up to four CRISPR-Cas systems, referred to herein as CR1 through to CR4. CR1 and CR3 are classified as Type II-A and Type II-C systems, respectively, and are the most widely reported functional systems in this species (48). CR2 and CR4 are classified as Type III-A and Type I-E systems, respectively (1). The genomes of the 27 strains used in this study were scrutinized for the presence of CRISPR-Cas systems using CRISPRCasFinder, confirming the ubiquity of CR3 systems among the 27 analysed strains (Table 2). CR1 and CR2 were identified in 16 and 20 of the 27 genomes, respectively, while CR4 was present in just three genomes. Typically, CR2 systems are observed to incorporate/retain the lowest number of spacers, while CR3 and CR1 incorporate or retain considerably higher numbers of spacers perhaps reflecting the activity level of these systems. CR4 systems present in the genomes of strains 4134, 90728 and MM20 were observed to harbour 12, 18 and 20 spacers, respectively, which also suggests that these systems are functional and active (Table 2). The genetic regions specifying CR1, CR2 (where present) and CR3 CRISPR-Cas regions are located in conserved genomic positions in all analysed strains with the CR1 locus positioned between genes associated with orotidine biosynthesis, the CR2 locus (when present) located downstream of genes associated with septation ring formation and the CR3 locus located downstream of shikimate pathway-associated functions (Fig. 1A).

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<figure><img src="image_2.png" /><figcaption>**Fig. 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.</figcaption></figure>

**The *S. thermophilus* methylome is dominated by Type I systems**

The products of all identified ORFs of the genomes were compared by BLASTp alignments against the REBASE database (http://rebase.neb.com/rebase/rebase.html) to identify potential R/M system genes. Using this approach 78, 60, 14 and 18 predicted Type I, II, III and IV R/M systems were identified (albeit with apparent frameshifts in some) (Fig. 2; Supplementary Tables S3-S11). PacBio-derived genome sequence data sets were analysed to
identify DNA methylation motifs, and the genes for each methylase were matched with these
motifs wherever possible. In many cases, a predicted restriction enzyme-encoding gene was
located next to or close to an active methylase, indicating that a complete and active R/M
system is present. Among the 27 analysed genomes, 26 (all except strain 4067) were shown
to possess methylated motifs, which were solely represented by m6A base modifications. In
this manner, a total of 49 distinct methylated motifs were identified, among which 32 are
attributable to Type I, 11 to Type II and 6 to Type III R/M systems (Fig. 2; Supplementary
Tables S3, S4 and S5).

In addition to the 49 methylated motifs, each of which could be assigned to a specific R/M
system as based on alignments against the REBASE database, 29 Type I R/M system genes
were identified that do not have a corresponding methylated motif, often due to frameshift
mutations that rendered the system (perhaps temporarily) inactive (Supplementary Table S6).
Furthermore, five strains harbour an m5C Type II R/M system associated with a GATC
recognition sequence, two associated with a GCSGC recognition sequence and one with a
CCGG recognition sequence (Fig. 2; Supplementary Table S7 & S8). Methylation was not
detected for these systems in the present study, most likely owing to the technological
approach used (which does not accurately detect cytosine-specific methylations); however, it
is likely that these systems are functional. This highlights the presence of possible cytosine-
specific methyltransferases in this species in addition to the dominant adenine methylase
activity (Fig. 2). All known Type III R/M systems to date are described to (hemi)methylate an
adenine (m6A or m4C) (49). While the Type III R/M system associated with a CGCAT motif
was identified in the genome of ten strains, methylation of this motif was only observed in
the genomes of three strains i.e. CNRZ302, MM20 and UCCSt95 (Fig. 2). The apparent non-
functionality of this (and other) R/M systems appears to be due to observed frameshifts in
the associated gene(s) (Supplementary Table S8). Type IV systems do not encode a
methylase and typically cleave methylated or unusually modified (hydroxymethylated or
glucosyl-hydroxymethylated) bases (50) and may not be detected using the approach
applied in this study (Fig. 2 & Supplementary Table S10).

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<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2. Upper panel:** Identified R/M systems (both active systems and inactive systems/individual genes). Methylated bases within the detected motifs based on SMRT</figcaption></figure>
385 sequencing are indicated in blue text in the "motif" column. Predicted m5C (pink text) and
386 m6A (blue text) associated enzymes which were predicted but where methylation activity was
387 not detected in this study. Where more than one gene was identified, the number of such
388 genes is indicated by a number in the relevant box. In the "Enzyme" column, black text
389 indicates enzymes for which associated methylated motifs were identified, blue and pink text
390 indicates enzymes for which m6A and m5C recognition sequences, respectively, were
391 predicted but methylated motifs were not detected. **Lower panel:** Efficiency of plaquing
392 (EOP) of lactococcal phages in the presence (induced) or absence (uninduced) of Type I, Type
393 II or Type III R/M systems. In the uninduced state, there is no observable phage-resistance
394 while in the induced state, all three evaluated R/M systems were observed to elicit
395 substantial resistance against the evaluated phages 62606, 712, jj50, p2, c2 and sk1.
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397 **Restriction & modification systems are encoded by genes that occur in proximity to**
398 **CRISPR loci**

399 Overall, the 27 assessed S. thermophilus strains are predicted to encode between one and
400 five active R/M systems with an average of 3.4 R/M systems per strain. Type I R/M systems
401 were shown to be encoded by genes that occur at several distinct genomic locations. Among
402 the genetic determinants of the Type I systems analysed, the majority (24 of 33) are located
403 in a conserved location downstream of CR3 and to a lesser extent, in proximity to CR2. The
404 genes encoding identified Type II and Type III R/M systems occur in conserved locations
405 upstream or downstream of CR3. The Type IV systems are encoded by genes that typically
406 occur downstream of CR3 systems (in 17 out of 18 cases). In the case of strain MM20,
407 genomic rearrangements are evident although the genomic context of the predicted Type IV
408 remains the same as for the other strains while in contrast, the Type IV system of strain
409 CNRZ385 is present in a region rich in transposase elements, which may have facilitated
410 genomic inversions and which likely accounts for its unusual genomic location relative to the
411 other strains. While the vast majority of R/M systems identified in this study are
412 chromosomally-located, the enzymes responsible for two methylated DNA motifs appear to
413 be plasmid-encoded, i.e. S. thermophilus AVA116 (motif CCA(N7)TCG) and S. thermophilus
414 CNRZ1202 (motif GATC) (Fig. 2). In AVA116, a solitary hsdS specificity subunit is present in
415 the plasmid pAVA116A that is expected to interact with the chromosomal Type I methylase

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subunit to methylate its recognition sequence. Interestingly, PADLOC analysis did not identify this hsdS subunit, which may highlight the importance of methylation analysis and tailored searches for R/M system components to establish the complete methylome. The plasmid-associated Type II system of *S. thermophilus* CNRZ1202 is a complete system encoded by genes on the plasmid pCNRZ1202A (Table S2).

While the methylase functionality of the m6A systems was established through methylation profiling based on PacBio SMRT sequence data, it was decided to establish the functionality of representative cognate restriction enzymes in terms of providing phage resistance. This was achieved by cloning genes encoding a representative Type I (from *S. thermophilus* 4078; motif CCAYN<sub>5</sub>TGA), II (from *S. thermophilus* MM1; motif CAGRAG) or III (from *S. thermophilus* St95; motif ACAGC) R/M system in the low copy vector pPTPi, after which the resulting constructs were introduced into the R/M-free lactococcal strain *L. cremoris* VES7862 by electroporation. Molecular tools to modify the genomes of *S. thermophilus* are not as well developed as for some bacterial species (such as *L. cremoris*), while the genetic amenability of individual *S. thermophilus* strains is also unpredictable and often poor. Therefore, functional validation of the selected R/M systems was performed in *Lactococcus cremoris* due to the availability of genetic tools to generate a R/M-free derivative of the test strain and the similar GC % content of *Lactococcus* and *S. thermophilus*. The R/M system-carrying strains were evaluated for their ability to reduce the efficiency of plaquing of the lactococcal skunaviruses sk1, p2, jj50 and 62606 and the *Ceduovirus* c2. All three heterologously expressed R/M systems elicited a clear phage-resistance phenotype (three to five log reduction in E.O.P.) against all tested phages confirming the functionality of the selected R/M systems' restriction enzymes in addition to the defined methylase function (Fig. 2; Table 3). Cross-species functionality of R/M (and other phage-resistance) systems between mesophilic lactococci and thermophilic dairy streptococci has previously been reported. Specifically, the Type II R/M system LLall (which recognises a GATC target sequence) identified on the lactococcal plasmid pSRQ700 was found to be functional at 42 °C in *S. thermophilus* when provided *in trans* on a suitable replicating vector (51).

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447 **S. thermophilus defence islands**

448 Several studies have reported the co-location of phage-resistance systems in so-called
449 "Defence Islands" in both Gram-negative and Gram-positive bacteria (11, 15, 17).

450 Furthermore, analysis of the CRISPR-Cas and R/M systems in this study, identified that these
451 are most often located in conserved locations in dairy streptococcal genomes and in relative
452 proximity between certain CRISPR-Cas and R/M systems. Therefore, it was considered that
453 these regions constitute Defence Islands in *S. thermophilus* and represent locations to
454 identify additional phage-resistance systems. Since CRISPR-Cas systems are always present in
455 *S. thermophilus* genomes, we chose to use the CR1, CR2 and CR3 regions as genomic
456 beacons for these Defence Islands. Herein, the Defence Islands are named in accordance with
457 the CR region with which they coincide and will be discussed in order of appearance in the
458 genomes of the analysed strains, i.e. CR3-Defence Island, CR2/4-Defence Island and CR1-
459 Defence Island (Fig. 1A). CR4 regions were identified in the genomes of just three strains and
460 these are located in relative proximity to CR2 systems and would thus be considered part of
461 the CR2/4-Defence Island. Using the outputs of the PADLOC analysis mentioned above, the
462 location of the identified non-CRISPR and non-R/M phage-resistance systems was evaluated
463 in the context of these three proposed Defence Islands. Additionally, the prophage regions
464 identified in the analysed genomes were evaluated for their proximity to CR-Defence
465 Islands. Among the identified prophage regions, eight are in a similar relative position on
466 the genome between the CR3- and CR2/4-Defence Islands. These prophage regions are
467 "questionable" or "incomplete" prophage regions and primarily incorporate transposases, an
468 integrase-encoding gene, a structural protein-encoding gene and genes predicted to encode
469 nucleoside phosphorylases across regions spanning ~37-41 kb. Also included in this cohort
470 is one predicted intact prophage (in strain R1's genome) although manual inspection did not
471 identify an obvious morphogenesis module rendering it unlikely to form infective phage
472 particles. The location of these prophage regions (although seemingly cryptic prophage
473 regions) between the CRISPR loci combined with the density of transposase-encoding genes
474 in this region highlights the likely genomic plasticity of this region among *S. thermophilus*
475 strains. The remaining four prophage regions are in a variety of genomic positions across
476 their hosts' genomes without identifiable phage-resistance systems identified within them.
478 **CR3-Defence Island- a hotspot of abortive infection anti-phage systems**

479 CR3-Defence Island is the largest of the three defence islands with an average size of 74.5 kb
480 (ranging from 51.0-128.5 kb) incorporating on average 77 predicted genes (ranging from 62-
481 131 genes). This island is flanked by a conserved gene cluster associated with the shikimate
482 pathway at the 5' end and a conserved heat shock protein-encoding gene at the 3' end.

483 Between the genes encoding these functions are a plethora of (putative) anti-phage systems
484 including CR3 (all strains) as well as Type I, Type II and/or Type IV R/M systems in conserved
485 locations relative to CR3 (Fig. 1B). Within this region, there are several highly conserved
486 genes (>90% sequence similarity) with predicted functions including manganese transport,
487 proteases, biotin synthesis and histidine protein kinase, among others. Approximately 25
488 genes of mostly unknown function represent the variable gene content in the overall CR3-
489 Defence Island. Among these variable genes of the CR3-Defence Island, PADLOC analysis
490 identified genes encoding predicted AbiD, GAO_19, Kiwa and Gabija in a small number of
491 strains (Table 4; Fig. 1B & Fig. 3A).

492 Defence-associated sirtuins (DSRs) have been reported to deplete NAD+ through the
493 NADase activity of SIR2 (called GAO_19 systems) and exhibit an Abi phenotype (52). Abi
494 phenotypes are typified by strain collapse in the presence of phages at high multiplicities of
495 infection (MOI) but not where low MOIs are applied. For their activity, GAO_19 systems
496 require a second protein, which differs depending on the host species, i.e. the DNA
497 translocase HerA or pAgo. Bacterial DSRs of *Bacillus subtilis* have recently been
498 demonstrated to recognize the phage tail tube leading to the activation of SIR2-mediated
499 NAD+ hydrolysis (53). The chromosomes of *S. thermophilus* STR1, CNRZ385, ST55, 4145 and
500 4021 harbour homologues of the SIR2-dependent NAD+ depletion system encoded by
501 *sir2/herA* (Table 4). The encoded SIR2/HerA system of CNRZ385 and 4021 are almost
502 identical to each other (SIR2: 100 % identity; HerA: 99 % identity over 88 % of the protein
503 sequence) as are those of strains 4145 and ST55 (SIR2: 96.3 % identity; HerA: 97.8 % identity).
504 The associated gene pair is in a similar position in the genome of each of the carrying strains
505 (Fig. 1A & B). Conversely, the SIR2/HerA system of STR1 displays markedly reduced sequence
506 similarity to those of the other four strains. The GAO_19 SIR2/HerA systems are observed to
507 be inserted between (R1, CNRZ385 and 4021) or downstream of (ST55 and 4145) R/M
508 systems in CR3-Defence Island (Fig. 1B & Fig. 3A). The GAO_19 *sir2/herA* gene pairs of R1
and 4021 were cloned in pNZ44 as representatives of the two genotypes of this system and transformed into lactococcal and streptococcal host strains to establish their ability to confer phage-resistance. GAO_19<sub>4021</sub> provides resistance against sk1 and a moderate level of protection against SW13 and c2, while GAO_19<sub>STR1</sub> provides up to three logs of protection against three of the tested streptococcal phages (Fig. 3B).

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A

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B

<table><thead><tr><th rowspan="2">Phage assay<br>host</th><th colspan="4">S. thermophilus</th><th colspan="2">L. cremoris</th></tr><tr><th>SW13</th><th>STP1</th><th>P738</th><th>SW16</th><th>sk1</th><th>c2</th></tr></thead><tbody><tr><td>Construct/system</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GAO19_STR1</td><td>§</td><td>§</td><td></td><td></td><td></td><td></td></tr><tr><td>GAO19_4021</td><td></td><td></td><td></td><td></td><td>§</td><td>§</td></tr><tr><td>Gabija_4145</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Kiwa_90730</td><td></td><td>§</td><td>§</td><td>§</td><td>§</td><td>§</td></tr><tr><td>Hachiman_ST19</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PD-T4-6_4078</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PD-T4-6_4078_U</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SoFic_MM20</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dodola_90729</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AbiD_ST55</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AbiD_ST55_U</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AbiD_4021</td><td>§</td><td>§</td><td></td><td></td><td></td><td></td></tr><tr><td>AbiD_4021_U</td><td></td><td>§</td><td></td><td></td><td></td><td></td></tr><tr><td>AbiD_4078</td><td>§</td><td></td><td></td><td></td><td>§</td><td>§</td></tr><tr><td>AbiD_4078_U</td><td></td><td></td><td></td><td></td><td>§</td><td>§</td></tr><tr><td>AbiE_90730</td><td>§</td><td>§</td><td></td><td></td><td></td><td></td></tr><tr><td>AbiE_90730_U</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

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<caption>**Fig. 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.</caption>

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SIR2 proteins are well characterized in eukaryotes and have been implicated in transcriptional silencing, cell cycle progression and genome stabilization functions (54). All
SIR2 homologues studied to date possess NAD-dependent deacetylase activity highlighting the central role of this activity to the functionality of the system (55–57). The structures of the GAO_19 associated sirtuin-dependent systems identified in this study (ST4021-0687 (SIR2) and ST4021-0688 (HerA)) were predicted with AlphaFold2 (Fig. 4A-D; Supplementary Fig. S4E-G). SIR2<sub>4021</sub> has a compact α/β-fold. Dali analysis of SIR2<sub>4021</sub> identified hits with NAD-dependent deacetylase (PDB 4twi-A) and a transcriptional regulatory protein of the SIR2 family (PDB 1ici). The predicted structure of SIR2<sub>4021</sub> superimposes well on the SIR2 of PDB 1ici (Fig. 4B). HerA<sub>4021</sub> is a three-domain protein that exhibits structural similarity to and superimposes well on HerA (PDB 4d2i; Fig. 4C, 4D) based on Dali analysis. Structure prediction of a hexamer of HerA<sub>4021</sub> produces a model with a high confidence level similar to HerA (PDB 4d2i). In *Thermus thermophilus* this protein is defined as possessing ATPase activity whose structure comprises a conically-shaped, double hexamer ring with a central pore, consistent with our predictions (Fig. 4E & 4F) (58).
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<caption>**Fig. 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=4.2Å). (E) molecular surface representations (side and top views) of 4021-0688 hexamer compared to (F) the molecular surface of the HerA ATPase from *S. 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).</caption>
552 Gabija was first identified in *Bacillus cereus* and was observed to be active against Myo-, 553 Sipho- and Podophages and was found in 8.5 % of microbial genomes tested (17). This is a 554 two protein sensor-effector system incorporating GajA, an ATPase with DNA nicking activity, 555 and GajB, a helicase, which together form a ~500 kDa DNA-degrading complex (59). GajB 556 senses DNA termini leading to nucleotide hydrolysis and the concomitant nucleotide 557 depletion culminates in cellular abortion (14, 60). In contrast to the broad spectrum of 558 activity of previously described Gabija systems, no significant phage-resistance was elicited 559 against the tested phages by Gabija₄₁₄₅ with the exception of a very modest antiphage 560 activity (0.5-1 log reduction in EOP) against STP1 and SW16 (Fig. 3B). The Gabija system 561 identified in the genome of 4145 is encoded by *ST4145_0685* and *ST4145_0686*. AlphaFold 562 prediction of the Gabija proteins 3D structure is consistent with previous functional analysis 563 in which GajA yielded a three-domain protein that assembles as a dimer (Supplementary Fig. 564 S1A, B, S4H) and with Dali hits with an ATP-dependent endonuclease (PDB 6p74) (Fig. S1C). 565 GajB₄₁₄₅ was predicted as a compact α/β-protein that yielded Dali hits against DNA helicases 566 (PDB 4c2u; 4c30; 4c2t) (Supplementary Fig. S1D, S1E, S4I). Gabija is a seemingly rare system 567 among *S. thermophilus* strains as BLASTn analysis of *ST4145_0685* and *ST4145_0686* against 568 the NCBI database identified just a single homologue of this gene pair in *S. thermophilus* 569 ST64987 (E-value 0.00).

570 Kiwa incorporates *kwaA/kwaB* (Fig. 3A) and is present in 1.8 % of microbial genomes 571 evaluated previously (17). The encoded system is described as a membrane-associated 572 system that is triggered by the inhibition of host RNA polymerase and interferes with phage 573 replication through a RecBCD-dependent pathway (61). While KwaB exerts an anti-phage 574 effect independently of KwaA, it is believed that KwaA is required for its controlled 575 expression (61). KwaB is predicted to be a two-domain protein with an α/β-N-terminal 576 domain and an all α-helical C-terminal domain based on AlphaFold analysis (Supplementary 577 Fig. S1F, S4J). KwaB is predicted to dimerize primarily through its N-terminal domain (lower 578 parts of structure in Supplementary Fig. S1G, S4L). KwaA was predicted as an extended 579 mainly α-helical protein (Supplementary Fig. S1H, S4K) and produced hits against the *E. coli* 580 stress protein YciF (PDB 2gs4; Supplementary Fig. S1I) (62). Kiwa₉₀₇₃₀ exhibits a broad 581 spectrum with strong anti-phage activity against lactococcal phages sk1 and c2 (5- and 4-log
reduction of plaquing ability, respectively) and rather modest (1 log) resistance and plaque size reduction against tested streptococcal phages (Fig. 3B & Table S12).

AbiD-encoding homologues were identified in the chromosome of eight strains, i.e. S. thermophilus ST1A, CNRZ302, CNRZ760, 90728, 90729, ST55, 4021 and 4078 based on BLASTn searches in the NCBI database using default settings (Fig. 3A). BLASTp comparisons of the eight AbiD proteins identified three distinct sets based on the level of sequence similarity, i.e. AbiD encoded by ST55, 90728, 90729, and CNRZ760; AbiD of CNRZ302 and 4078; and AbiD of 4021 and ST1A. Those identified in strains ST55, 90728, 90729 and CNRZ760 are all associated with a large genomic insertion flanked by several transposase-encoding genes that essentially doubles the size of this Defence Island in the genomes of these strains. The insertion is conserved across these four strains and includes genes with predicted products including bacteriocin biosynthesis, SOS response protein UmuC and heavy metal resistance functions. The AbiD proteins encoded by members of each of the three groups are identical within the groups while between groups, the sequence identity drops to ~25 % across approximately half of the compared amino acid sequence. Six of the eight AbiD-encoding homologues are located within CR3-Defence Island. In the genome of CNRZ302, the AbiD-encoding gene is directly upstream of a Type II R/M system (Fig. 3A).

Alphafold2 analysis of representative AbiD proteins (those encoded by ST55, 4078 and 4021) established significant structural conservation of these proteins despite their low levels of sequence identity (Supplementary Fig. S2A-C, S4A-C). Furthermore, superimposition of these three proteins with a lactococcal AbiD protein (Supplementary Fig. S2D, S4D). highlights the maintenance of structural similarity of AbiD across these distinct species despite amino acid sequence similarities of approximately 5 % (Fig. S2D-F). Furthermore AbiD-ST55, as an example, shares structural homology with the core of PDB ID 6iv8 (residues 163-330 and 491-576), a Cas13d ribonuclease (Supplementary Fig. 2E). BLASTn analysis of AbiD-encoding genes revealed identical homologues in the genomes of nineteen strains of S. thermophilus in the NCBI database, highlighting the broad presence of this system among dairy streptococci. The evaluated AbiD systems were observed to provide low to moderate levels of protection against the streptococcal phages, while only AbiD₄₀₇₈ was observed to exert an effect against assessed lactococcal phages (Fig. 3B).
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CR2/4 and CR1-Defence Islands rely on innate and adaptive immune systems

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The CR2/4-Defence Island is demarcated by genes associated with orotidine biosynthesis flanking a variably present CR2 region and in two strains a CR4 region. This island spans approximately 17.5 kb (ranging from 15.9 – 18.7 kb) and typically contains ~20 open reading frames among which an average of five predicted genes have no assigned functions. This region is quite conserved among *S. thermophilus* strains although strain-specific genes are present among the unassigned genes. While CR2 and CR4 are less prevalent when compared to CR3 and CR1, the CR2/4-Defence island remains an interesting region to analyse for the presence of novel anti-phage systems since Type III R/M systems are always (and Type I R/M systems occasionally) located upstream of this region. Furthermore, the region upstream of the CR2/4-Defence Island is rich in IS-elements and transposase-encoding genes and predicted prophage regions in eight of the analysed genomes (as described above) while the highly variable *eps* locus (which encodes genes associated with exopolysaccharide biosynthesis) is located downstream of this island. The *eps* loci of *S. thermophilus* have previously been demonstrated to be genetically diverse and often harbour several transposase-encoding genes that are believed to contribute to the diversification of these clusters (63–65).

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The CR1-Defence Island is a highly conserved genomic region spanning approximately 21 kb (range: 20-22 kb) and represents the region which harbours CR1 (17 of 27 analysed genomes). CR1-defence Island is also associated with the presence of Type I or Type II R/M systems in some strains genomes. CR1-Defence Island is flanked by genes which encode septation and glutamine synthesis. This Defence Island incorporates approximately 20 genes, of which on average eight have no assigned function (Fig. 1). Interestingly, in five (i.e. CNRZ887, CNRZ1151, 90730, 4052 and 4147 (Table 4, Fig. 3A) of the ten strains that do not possess a CR1 region, the same relative genomic position is occupied by an AbiEi/Eii system, which is identical among these five strains (Fig. 1). HHPred analysis established structural homology to toxin and antitoxin proteins (99 % probability; PDB 6Y5U_A and 6Y8Q_B), while the 3D structure prediction of AbiEi was also found to superimpose well with the *S. agalactiae* antitoxin (PDB 6y5u) (Fig. 5A & 5B). AlphaFold2 analysis of the product of ORF 90730_1387 revealed that the carboxy-terminus of the protein (AbiEii) is likely truncated since these toxins are bilobial enzymes with an active site located in a crevice between the
two lobes encoded by the amino and carboxy-termini (Fig. 5C & D). Besides, the 5-stranded β-sheet of 6j7n is also lacking. Therefore, while the catalytic residues of the active site are present (Fig. 5D), the truncation of the protein is likely to affect the functionality of the system. This is consistent with plaque assay results where there is a limited impact on the plaquing efficiency of the phages tested in this study was observed (Fig. 3B).

<figure><img src="image_12.png" /><figcaption>**Fig. 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=2.7Å). (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=1.8Å). The red dot line indicates the catalytic crevice and the red and blue dots the positions of the catalytic residues in AbiEii and 6j7n.</figcaption></figure>

**Strain-specific acquisitions enhance the anti-phage repertoire of S. thermophilus**

While most of the anti-phage systems identified in this study are associated with the three CR-Defence Islands, genomes of individual strains were predicted to harbour unique anti-phage systems including SoFic, Hachiman, PD-T4-6 and Dodola. These systems can broadly be classified under the abortive infection category of phage resistance systems and their corresponding genes are positioned as standalone units at diverse locations across the
663 genome. In functional assays, these systems do appear to elicit a minimal impact on phage
664 resistance against the tested phages although it cannot be precluded that they operate
665 synergistically with other systems in the host or that they are active against other phages
666 beyond those evaluated in this study (Fig. 3B) (66).

667 SoFic systems are reported to be widespread in bacterial genomes and a Class II Fic domain-
668 encoding system was recently validated to be functional against coliphage T5 (15). These
669 systems are typified by those with so-called "Fic" domains with predicted protein AMPylase
670 activity. Structural prediction of SoFicMM20 in the present study yielded a similar functional
671 prediction (Supplementary Fig. S3A & S3B, S5F).

672 PADLOC analysis identified two identical copies of a predicted SoFic-encoding gene in the
673 genome of MM20 (Table 4). These genes occur at two disparate locations on the genome of
674 MM20, i.e. orf0248MM20 and orf1073MM20. This system is relatively rare among S. thermophilus
675 strains with just three almost identical (98.55 % sequence identity) homologues of this gene
676 identified in the NCBI database. AlphaFold predicts SoFic as an α-helical protein
677 (Supplementary Fig. S3A, S5F). Dali reports hits of SoFic with a domain of 6i7g, an Adenosine
678 Monophosphate-Protein Transferase Ficd with strong statistics (Z= 26.2; rmsd=1.8)
679 (Supplementary Fig. S3B, C).

680 Hachiman was first identified in *Bacillus cereus* and incorporates two proteins termed HamB,
681 which is a helicase and HamA, which is a nuclease. Hachiman has been reported to be
682 present in 3.4 % of microbial genomes evaluated (17). BLASTn analysis of *St19_0447* and
683 *St19_0448* identified a single homologue of these genes in the *S. thermophilus* strain EU01
684 with 100 % nucleotide sequence identity (E-value 0.00). AlphaFold2 predictions of HamA and
685 HamB concur with previous functional studies. HamA is predicted as a compact α/β protein
686 (Supplementary Fig. S3D; S5C) and with hits in the Dali server against the EC869 toxin (4g6u),
687 a Zn(2+)-dependent DNase (Supplementary Fig. S3E). Superimposition of HamA on 4g6u
688 yielded weak statistics (Z=5.7; rmsd=3.7Å). HamB is predicted as a multidomain α/β protein
689 (Supplementary Fig. S3F; S5D). Dali reports a strong hit for HamB with PDB 4u4c, an ATP-
690 dependent RNA helicase (Z=30.7; rmsd=3.7 Å). SoFicMM20 and HachimanST19 provide
691 resistance against P738 (P738 group namesake) and SW16 (987 group member) (Fig. 3B). It is
692 noteworthy that members of the P738 and 987 groups of dairy streptococcal phages are
693 relatively rarely encountered in dairy fermentations compared to members of the
694 *Brussowvirus* and *Moineauvirus* genera. Therefore, we hypothesize that the consistent
695 exposure of *Brussowvirus* and *Moineauvirus* members to dairy streptococcal strains and their
696 phage-resistance systems has provided them an opportunity to overcome many of the
697 identified phage-resistance systems through mutations or insertion/deletion events in
698 contrast to members of the rare P738 and 987 streptococcal phage groups.

699 PD-T4-6 is one of several *E. coli* derived systems that is proposed to have DNA
700 binding/cleavage activity although the specific mode of action remains to be elucidated (18).
701 AlphaFold2 prediction of full-length PD-T4-6 is rather complex (Supplementary Fig. S3H,
702 S5E). The N-terminal region (residues 1-278; red box) displays a compact structure (Fig. S3H,
703 I) and Dali server analysis of the N-terminal domain reports a clear hit (Z=34.8; rmsd=1.5 Å)
704 with 4eqm (Fig. S3J), a serine/threonine protein kinase. This, and two subsequent modules, in
705 tandem are formed of three antiparallel β-strands and one α-helix (Fig.8H, K), a structure
706 retrieved by Dali in PDB ID 3py9 (Z=13, rmsd=0.7 Å), a serine/threonine protein kinase
707 binding module (Fig. S3L). Interestingly, the PD-T4-6 encoding gene is present in all analysed
708 *S. thermophilus* genomes and was associated with a very mild phage-resistance phenotype in
709 *Lactococcus* (Fig. 3B).

710 Dodola is a two gene system (*dolA, dolB*) that has been shown to exert anti-phage activity
711 against *Bacillus subtilis* phage SPP1 (15). Here, a single representative of this system was
712 identified in the genome of strain 90729 (Table 4). DolA is of unknown function while DolB is
713 predicted to possess a ClpB domain, which is typically associated with ATPase activity. The
714 genes encoding Dodola<sub>90729</sub> are located at the distal end of the genome of this strain and
715 while it is not directly proximal to other phage-resistance systems, a Type II R/M system
716 (GATC) is encoded by two genes that are located further downstream, which may be
717 suggestive of a Defence Island. This is further supported by the variable occurrence of R/M
718 systems in this genomic region in other strains. AlphaFold2 analysis of Dodola A (DolA)
719 reports a three-domain protein: an α/β N-terminal domain, an α/β middle domain and an α-
720 helical C-terminal domain (Supplementary Fig. S3M). Dali reports hits of DolA with ATP-
721 dependent Clp protease ATP-binding subunits 7xbk (Supplementary Fig. S3N). To note, DolA
722 is predicted with high confidence to assemble as a hexamer, as does 7xbk (Supplementary
723 Fig. S3O, S5I). DolB is predicted as a two domains protein associating a N-terminal α-helical
domain to a α/β-C-terminal domain (Supplementary Fig. S3P, S5H). Dali reports weak hits with several proteins involved in DNA/RNA binding/modification, which does not allow a precise functional assignment.

## Distribution of anti-phage systems in S. thermophilus

The presence and distribution of anti-phage systems in S. thermophilus genome sequence data available in the RefSeq database was analysed using DefenseFinder to (a) establish if the 27 genomes of our strain collection were reflective of the species overall and (b) to identify additional systems beyond those identified in the genomes sequenced as part of the present study. Using "Streptococcus thermophilus" as a search term in the RefSeq database option of DefenseFinder, CRISPR-Cas and R/M systems were confirmed to be the globally dominant anti-phage systems in this species among 462 RefSeq entries (199 Cas and 187 R/M systems identified; Fig. 6A). Beyond these dominant systems, a significantly smaller proportion of Abi systems including AbiD/D1, AbiH, AbiE and Gabija were also identified. Interestingly, while many systems are similar to those identified in the genomes of the 27 strains analysed in this study (Gabija, Kiwa, Dodola, AbiD, AbiE), there are a small number (and low number of occurrences) of additional predicted systems such as Borvo, PrrC, PD-lambda-1 and PD-T7-2. This highlights that while this species readily harnesses CRISPR-Cas and R/M systems, strain-specific acquisitions may confer an advantage on the hosting strain. As the number of genome sequences available within this species increases, it will be possible to establish the true extent of the diversification of the phage-resistome of dairy streptococci.

## Conclusions

In the present study, the phage resistance landscapes of 27 S. thermophilus strains were elucidated to establish the diversity and functionality of anti-phage systems in this species (Fig. 6B). The innate and adaptive bacterial immune systems represented by R/M and CRISPR-Cas systems, respectively, are equipped to deal with "slow"-infecting phages or chronic infections (67). However, to cope with acute infections elicited by so-called "fast"-infecting phages, S. thermophilus has acquired abortive infection (Abi) systems and systems that are described to directly or indirectly activate abortive infection. These systems include AbiD, AbiE, SoFic, Dodola, Hachiman, Kiwa and GAO_19.
This collective of biological hurdles to deal with "slow" and "fast"-infecting phages (or chronic and acute infections) provides *S. thermophilus* with a strain level and tailored series of defences to diminish the impact of phages present in their ecological niche. The strain level diversity of anti-phage systems (Fig. 6B) may present a benefit at community level to ensure that *S. thermophilus* persists despite the pervasive presence of phages in dairy environments. Furthermore, since *S. thermophilus* has adapted to the dairy niche similar to *L. lactis/cremoris*, it is perhaps of broader benefit to the larger community of microorganisms that may be present in the same ecosystem. While receptor diversity/modification may act as the first line of defence against invading phages (25, 68), the role of antiphage systems in limiting the impact on surrounding populations of cells and in *Pseudomonas*, higher phage-resistance was correlated in strains whose genomes possessed a higher number of antiphage systems (69). Here, we identified nine distinct systems in addition to R/M and CRISPR-Cas systems that have not previously been functionally evaluated in *S. thermophilus*. Intriguingly, and in contrast to other bacterial genera, four of these systems (Hachiman, PD-T4-6, SoFic and Dodola) do not appear to be part of any obvious Defence Island. However, the identification of these "lone" phage-resistance systems may form an anchor to identify additional phage-resistance systems and/or Defence Islands in this species in the future. In addition, several so-called "phage defence candidate" (PDC) genes were identified in the genomes of these strains indicating that the potential reservoir of phage-resistance systems may be far greater than we currently realize.

Several of the identified systems were observed to be functional, not only in *S. thermophilus*, but also in *L. cremoris*. This cross-genus functionality provides strong evidence of the interactions of these lactic acid bacteria in agricultural and/or food production systems providing opportunities for mutually beneficial DNA transfer events. The current study reports the dedication of up to 2.5 % of *S. thermophilus* genomes to anti-phage activity. It is tempting to suggest that this number represents the "tip of the iceberg" and is likely significantly higher if we consider the newly identified systems in the present study forming the basis of much larger Defence Islands. Therefore, this study provides the foundation for systematic searches for phage-resistance systems within the identified Defence Islands and potential anchor regions for new Defence Islands in *S. thermophilus*.
A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /><figcaption>**Fig. 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</figcaption></figure>
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.

795

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808

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810

811
812 **Data Availability**

813 All coordinates of predicted structures are deposited in the open data repository Zenodo
814 (DOI 10.5281/zenodo.11221718). All RM-associated methylome and functionality data is
815 available through REBASE (http://rebase.neb.com/rebase/).

816

817 **Supplementary Data Statement**

818 Supplementary Data are available at NAR online. Supplementary Fig. S1 and Fig. S2: pLDDT
819 values and predicted aligned errors (PAE) plots of predicted structures.

820 **Funding**

821 This publication has emanated from research conducted with the financial support of Science
822 Foundation Ireland under grant numbers 20/FFP-P/8664, 12/RC/2273-P2 and 17/SP/4678,
823 which is co-funded by dsm-firmenich, Taste, Texture & Health. For the purpose of open
824 access, we have applied a CC BY public copyright license to any author accepted manuscript
825 version arising from this submission.

826 This work was performed in part using HPC resources from GENCI-IDRIS (Grant 2023-
827 AD010714075).

828 **Conflict of Interest**

829 PdW and NvP are employed by DSM-Firmenich. AF and RJR are employed by New England
830 Biolabs. The remaining authors declare that the research was conducted in the absence of
831 any commercial or financial relationships that could be construed as a potential conflict of
832 interest.

833 **Acknowledgements**

834 We acknowledge UCSF ChimeraX for molecular graphics that is developed by the Resource
835 for Biocomputing, Visualization, and Informatics at the University of California, San Francisco,
836 with support from National Institutes of Health R01-GM129325 and the Office of Cyber
837 Infrastructure and Computational Biology, National Institute of Allergy and Infectious
838 Diseases.
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TABLE AND FIGURE LEGENDS

<table><caption>Table 1. *S. thermophilus* strains applied in this study and their associated genome features</caption><thead><tr><th>S. thermophilus strain</th><th>Genome size (Mbps)</th><th>GC % content</th><th>#CDS</th><th>Phage-resistance regions of genome [kb (%)]</th><th>Genbank accession no.</th></tr></thead><tbody><tr><td>4021</td><td>1.861</td><td>39.07</td><td>1937</td><td>30.599 (1.6)</td><td>CP065384</td></tr><tr><td>4052</td><td>1.782</td><td>39.06</td><td>1883</td><td>23.994 (1.4)</td><td>CP065493</td></tr><tr><td>4067</td><td>1.821</td><td>39.01</td><td>1920</td><td>17.792 (1)</td><td>CP065476</td></tr><tr><td>4078</td><td>1.837</td><td>39.08</td><td>1944</td><td>28.536 (1.6)</td><td>CP065496</td></tr><tr><td>4134</td><td>1.855</td><td>39.08</td><td>1933</td><td>44.783 (2.4)</td><td>CP065477</td></tr><tr><td>4145</td><td>1.838</td><td>39.05</td><td>1931</td><td>35.111 (1.9)</td><td>CP065492</td></tr><tr><td>4147</td><td>1.797</td><td>39.08</td><td>1891</td><td>27.035 (1.5)</td><td>CP065504</td></tr><tr><td>90728</td><td>1.872</td><td>39.07</td><td>1959</td><td>41.833 (2.2)</td><td>CP065479</td></tr><tr><td>90729</td><td>1.877</td><td>39.14</td><td>1953</td><td>34.582 (1.8)</td><td>CP065480</td></tr><tr><td>90730</td><td>1.796</td><td>39.05</td><td>1883</td><td>25.360 (1.4)</td><td>CP065481</td></tr><tr><td>AVA1121</td><td>1.829</td><td>39.07</td><td>2003</td><td>29.506 (1.6)</td><td>CP065488</td></tr><tr><td>AVA116</td><td>1.737</td><td>39.21</td><td>1821</td><td>29.506 (1.7)</td><td>CP065498</td></tr><tr><td>CNRZ1151</td><td>1.794</td><td>39.05</td><td>1890</td><td>26.654 (1.5)</td><td>CP065483</td></tr><tr><td>CNRZ1202</td><td>1.79</td><td>39.09</td><td>1880</td><td>17.044 (1)</td><td>CP065506</td></tr><tr><td>CNRZ1575</td><td>1.823</td><td>39.21</td><td>1943</td><td>17.044 (0.9)</td><td>CP065490</td></tr><tr><td>CNRZ302</td><td>1.859</td><td>39.09</td><td>1975</td><td>33.663 (1.8)</td><td>CP065489</td></tr><tr><td>CNRZ385</td><td>1.897</td><td>39.07</td><td>1995</td><td>33.647 (1.8)</td><td>CP065495</td></tr><tr><td>CNRZ760</td><td>1.871</td><td>39.06</td><td>1959</td><td>31.448 (1.7)</td><td>CP065482</td></tr><tr><td>CNRZ887</td><td>1.78</td><td>39.13</td><td>1854</td><td>23.694 (1.3)</td><td>CP065491</td></tr><tr><td>MM1</td><td>1.843</td><td>39.1</td><td>1927</td><td>24.641 (1.4)</td><td>CP065484</td></tr><tr><td>MM20</td><td>1.942</td><td>38.81</td><td>2023</td><td>41.921 (2.2)</td><td>CP065485</td></tr><tr><td>ST128</td><td>1.854</td><td>39.12</td><td>1929</td><td>38.379 (2)</td><td>CP065500</td></tr><tr><td>ST19</td><td>1.863</td><td>39.01</td><td>1929</td><td>40.001 (2.2)</td><td>CP065487</td></tr><tr><td>ST1A</td><td>1.869</td><td>39.06</td><td>1947</td><td>29.721 (1.6)</td><td>CP065384</td></tr><tr><td>ST55</td><td>1.865</td><td>38.95</td><td>1964</td><td>36.626 (2)</td><td>CP065502</td></tr><tr><td>UCCSt95</td><td>1.794</td><td>39.19</td><td>1868</td><td>37.323 (2.1)</td><td>CP101646</td></tr><tr><td>R1</td><td>1.859</td><td>39.02</td><td>1951</td><td>26.701 (1.4)</td><td>CP065486</td></tr></tbody></table>

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1055

1056
<table><caption>**Table 2.** CRISPR-Cas profiles* and prophage characteristics of *S. thermophilus* strains with number of spacers/prophages indicated. Grey shaded boxes indicate absence of features.</caption><thead><tr><th>Sth strain</th><th>CR1</th><th>CR2</th><th>CR3</th><th>CR4</th><th># Prophage regions predicted</th><th>Size (kb); location</th></tr></thead><tbody><tr><td>4021</td><td>14</td><td></td><td>16</td><td></td><td>1 questionable</td><td>40.7; 768552-809322</td></tr><tr><td>4052</td><td></td><td></td><td>24</td><td></td><td></td><td></td></tr><tr><td>4067</td><td></td><td></td><td>7</td><td></td><td>1 incomplete</td><td>32.6; 1649507-1682124</td></tr><tr><td>4078</td><td>20</td><td>1</td><td>36</td><td></td><td>1 intact</td><td>16.2; 960763-977025</td></tr><tr><td>4134</td><td>12</td><td>3</td><td>32</td><td>12</td><td>1 questionable</td><td>37.7; 761384-799143</td></tr><tr><td>4145</td><td>19</td><td>1</td><td>30</td><td></td><td>1 questionable</td><td>37.7; 752479-790239</td></tr><tr><td>4147</td><td></td><td>1</td><td>14</td><td></td><td></td><td></td></tr><tr><td>90728</td><td>16</td><td>4</td><td>10</td><td>18</td><td></td><td></td></tr><tr><td>90729</td><td></td><td>3</td><td>11</td><td></td><td></td><td></td></tr><tr><td>90730</td><td></td><td>1</td><td>29</td><td></td><td></td><td></td></tr><tr><td>AVA1121</td><td>16</td><td></td><td>9</td><td></td><td></td><td></td></tr><tr><td>AVA116</td><td></td><td>7</td><td>18</td><td></td><td></td><td></td></tr><tr><td>CNRZ1151</td><td></td><td>1</td><td>29</td><td></td><td></td><td></td></tr><tr><td>CNRZ1202</td><td></td><td></td><td>23</td><td></td><td></td><td></td></tr><tr><td>CNRZ1575</td><td>25</td><td></td><td>30</td><td></td><td></td><td></td></tr><tr><td>CNRZ302</td><td>17</td><td>1</td><td>24</td><td></td><td></td><td></td></tr><tr><td>CNRZ385</td><td>18</td><td>1</td><td>20</td><td></td><td>2 questionable</td><td>37.7; 766659-804403<br>23.7; 830040-853786</td></tr><tr><td>CNRZ760</td><td>35</td><td>1</td><td>28</td><td></td><td></td><td></td></tr><tr><td>CNRZ887</td><td></td><td>4</td><td>29</td><td></td><td></td><td></td></tr><tr><td>MM1</td><td></td><td>1</td><td>24</td><td></td><td></td><td></td></tr><tr><td>MM20</td><td></td><td>16</td><td>23</td><td>20</td><td></td><td></td></tr><tr><td>STR1</td><td>23</td><td></td><td>17</td><td></td><td>1 intact</td><td>37.1; 731756-768888</td></tr><tr><td>ST128</td><td>46</td><td>1</td><td>22</td><td></td><td>1 questionable</td><td>59.4; 23992-83425</td></tr><tr><td>ST19</td><td>17</td><td>3</td><td>37</td><td></td><td>1 questionable</td><td>37.8; 754480-792319</td></tr><tr><td>ST1A</td><td>16</td><td>3</td><td>16</td><td></td><td>1 questionable</td><td>40.7; 764773-805543</td></tr><tr><td>ST55</td><td>23</td><td>3</td><td>17</td><td></td><td></td><td></td></tr><tr><td>UCCSt95</td><td>28</td><td>1</td><td>27</td><td></td><td>1 questionable</td><td>37.6; 729101-766773</td></tr></tbody></table>

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<table><caption>**Table 3.** Efficiency of plaquing (EOP) of lactococcal phages in the presence or absence of Type I, Type II or Type III R/M systems</caption><thead><tr><th>VES7862<br>pPTPi<br>derivative</th><th>sk1</th><th>c2</th><th>p2</th><th>jj50</th><th>712</th><th>62606</th></tr></thead><tbody><tr><td>Empty vector control</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>4078_RM I<br>Uninduced</td><td>1.07 ± 0.12</td><td>1.02 ± 0.05</td><td>1.14 ± 0.06</td><td>1.11 ± 0.12</td><td>0.96 ± 0.08</td><td>1.10 ±<br>0.11</td></tr><tr><td>4078_RM I<br>Induced</td><td>4.0 × 10<sup>-5</sup> ±<br>1.1 × 10<sup>-5</sup></td><td>6.8 × 10<sup>-4</sup> ±<br>1.2 × 10<sup>-4</sup></td><td>2.6 × 10<sup>-5</sup> ±<br>9.5 × 10<sup>-6</sup></td><td>5.1 × 10<sup>-5</sup> ±<br>5.3 × 10<sup>-6</sup></td><td>5.9 × 10<sup>-4</sup> ±<br>1.7 × 10<sup>-4</sup></td><td>1.0 × 10<sup>-3</sup><br>± 2.9 ×<br>10<sup>-4</sup></td></tr><tr><td>MM1_RM II<br>Uninduced</td><td>1.11 ± 0.20</td><td>0.92 ± 0.10</td><td>1.27 ± 0.19</td><td>1.11 ± 0.11</td><td>0.92 ± 0.08</td><td>0.92 ±<br>0.10</td></tr><tr><td>MM1_RM II<br>Induced</td><td>2.1 × 10<sup>-4</sup> ±<br>5.5 × 10<sup>-5</sup></td><td>6.1 × 10<sup>-3</sup> ±<br>5.9 × 10<sup>-4</sup></td><td>1.1 × 10<sup>-4</sup> ±<br>3.1 × 10<sup>-5</sup></td><td>5.4 × 10<sup>-4</sup> ±<br>1.6 × 10<sup>-4</sup></td><td>9.7 × 10<sup>-4</sup> ±<br>1.8 × 10<sup>-4</sup></td><td>1.6 × 10<sup>-3</sup><br>± 4.1 ×<br>10<sup>-4</sup></td></tr><tr><td>ST95_RM III<br>Uninduced</td><td>1.06 ± 0.09</td><td>0.99 ± 0.07</td><td>1.03 ± 0.07</td><td>1.11 ± 0.12</td><td>0.97 ± 0.11</td><td>0.84 ±<br>0.12</td></tr><tr><td>ST95_RM III<br>Induced</td><td>1.1 × 10<sup>-4</sup> ±<br>3.1 × 10<sup>-5</sup></td><td>9.4 × 10<sup>-4</sup> ±<br>7.8 × 10<sup>-5</sup></td><td>1.6 × 10<sup>-5</sup> ±<br>4.8 × 10<sup>-6</sup></td><td>3.8 × 10<sup>-5</sup> ±<br>1.1 × 10<sup>-5</sup></td><td>6.3 × 10<sup>-4</sup> ±<br>6.7 × 10<sup>-5</sup></td><td>3.2 × 10<sup>-4</sup><br>± 8.4 ×<br>10<sup>-5</sup></td></tr></tbody></table>

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<table><caption>**Table 4.** Phage-resistance systems beyond CRISPR-Cas and R/M identified by PADLOC. Bold face text indicates the systems selected for functional evaluation.</caption><thead><tr><th>Strain</th><th>PADLOC identified phage-resistance systems</th><th>ORF number</th><th>Location on genome</th></tr></thead><tbody><tr><td rowspan="3">ST1A</td><td>AbiD</td><td>1639</td><td>1601461-1602441c</td></tr><tr><td>PD-T4-6</td><td>1341</td><td>1312938-1314809c</td></tr><tr><td>PDC-S29</td><td>1815</td><td>1760204-1760938c</td></tr><tr><td rowspan="2">UCCSt95</td><td>PDC-S07</td><td>0654</td><td>642989-643378</td></tr><tr><td>PD-T4-6</td><td>1285</td><td>1259864-1261735c</td></tr><tr><td rowspan="2">CNRZ302</td><td>AbiD</td><td>03920</td><td>760545-761435</td></tr><tr><td>PD-T4-6</td><td>1370</td><td>1309158-1311029c</td></tr><tr><td rowspan="2">CNRZ385</td><td>GAO_19 (SIR2, HerA)</td><td>0755, 0756</td><td>691015-693872</td></tr><tr><td>PD-T4-6</td><td>1417</td><td>1319589-1321460c</td></tr><tr><td rowspan="2">CNRZ760</td><td>AbiD</td><td>0656</td><td>641609-642619</td></tr><tr><td>PD-T4-6</td><td>1366</td><td>1312221-1314092c</td></tr><tr><td rowspan="2">CNRZ887</td><td>AbiEi/Eii</td><td>1349, 1350</td><td>1311759-1312675</td></tr><tr><td>PD-T4-6</td><td>1277</td><td>1239802-1241673c</td></tr><tr><td rowspan="2">CNRZ1151</td><td>AbiEi/Eii</td><td>1395, 1396</td><td>1334306-1335222</td></tr><tr><td>PD-T4-6</td><td>1328</td><td>1271059-1272930c</td></tr><tr><td rowspan="2">90728</td><td>AbiD</td><td>0633</td><td>621588-622598</td></tr><tr><td>PD-T4-6</td><td>1371</td><td>1323778-1325649c</td></tr><tr><td rowspan="2">90729</td><td>AbiD</td><td>0642</td><td>633131-634141</td></tr><tr><td>PD-T4-6</td><td>1342</td><td>1313302-1315173c</td></tr><tr><td rowspan="3">90730</td><td>Dodola (dolA, dolB)</td><td>1798, 1799</td><td>1746878-1748836c</td></tr><tr><td>AbiEi/Eii</td><td>1386, 1387</td><td>1336150-1337066</td></tr><tr><td>Kiwa (kwaB, kwaA)</td><td>0688, 0689</td><td>673224-674776</td></tr><tr><td rowspan="2">STR1</td><td>GAO_19 (SIR2, herA)</td><td>0691, 0692</td><td>681184-684217</td></tr><tr><td>PD-T4-6</td><td>1358</td><td>1311954-1313825c</td></tr><tr><td rowspan="2">ST19</td><td>Hachiman type I</td><td>0447, 0448</td><td>453847-457022</td></tr><tr><td>PD-T4-6</td><td>1322</td><td>1295405-1297276c</td></tr><tr><td rowspan="7">ST55</td><td>AbiD</td><td>0646</td><td>624941-625951</td></tr><tr><td>PDC-S58</td><td>0671</td><td>651314-652729</td></tr><tr><td>GAO_19 (SIR2, HerA)</td><td>0721, 0722</td><td>698303-701093</td></tr><tr><td>PDC-S06</td><td>0741</td><td>716435-718624</td></tr><tr><td>PDC-S61</td><td>0744</td><td>720509-721261</td></tr><tr><td>PD-T4-6</td><td>1380</td><td>1327282-1329153c</td></tr><tr><td>PD-T4-6</td><td>1338</td><td>1305964-1307835</td></tr><tr><td rowspan="2">ST128</td><td>GAO_19 (SIR2, HerA)</td><td>0687, 0688</td><td>681458-684306</td></tr><tr><td>AbiD</td><td>1632</td><td>1594135-1595115c</td></tr><tr><td rowspan="4">4052</td><td>PD-T4-6</td><td>1335</td><td>1305732-1307603c</td></tr><tr><td>PDC-S07</td><td>0664</td><td>643370-643759</td></tr><tr><td>PD-T4-6</td><td>1298</td><td>1239930-1241801c</td></tr><tr><td>AbiEi/Eii</td><td>1371, 1372</td><td>1309955-1310871</td></tr><tr><td rowspan="2">4078</td><td>AbiD</td><td>0760</td><td>741877-742767</td></tr><tr><td>PD-T4-6</td><td>1344</td><td>1286907-1288778c</td></tr><tr><td>4134</td><td>AbiEi/Eii</td><td>1337</td><td>1300554-1302425c</td></tr><tr><td rowspan="2">4145</td><td>Gabija (gajA, gajB)</td><td>0685, 0686</td><td>679404-682765</td></tr><tr><td>GAO_19 (SIR2/HerA)</td><td>0663, 0664</td><td>657103-659392</td></tr><tr><td rowspan="2">4147</td><td>PD-T4-6</td><td>1313</td><td>1283833-1285704c</td></tr><tr><td>AbiEi/Eii</td><td>1393, 1394</td><td>1337577-1338493</td></tr><tr><td>MM1</td><td>PD-T4-6</td><td>1317</td><td>1263929-1265800c</td></tr><tr><td rowspan="2">MM20</td><td>PD-T4-6</td><td>1333</td><td>1295884-1297755c</td></tr><tr><td>PD-T4-6</td><td>0620</td><td>604802-606673</td></tr><tr><td></td><td>Sofic</td><td>0248</td><td>248143-248967</td></tr><tr><td></td><td>Sofic</td><td>1073</td><td>1051268-1052092c</td></tr></tbody></table>
AVA116 PD-T4-6 1250 1210550-1212421c
AVA1121 PD-T4-6 1339 1276199-1278070c

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