
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39119896
10.1093/nar/gkae671
gkae671
AcademicSubjects/SCI00010
Molecular Biology
Discovery of antiphage systems in the lactococcal plasmidome
https://orcid.org/0000-0003-3803-4479
Grafakou Andriana School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

https://orcid.org/0000-0002-5369-8938
Mosterd Cas School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

https://orcid.org/0000-0003-1793-0629
Beck Matthias H School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

https://orcid.org/0000-0003-3423-7930
Kelleher Philip School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

https://orcid.org/0000-0003-1704-8759
McDonnell Brian School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

de Waal Paul P dsm-firmenich, Taste, Texture & Health, Center for Food Innovation, Delft 2613 AX, The Netherlands

van Rijswijck Irma M H dsm-firmenich, Taste, Texture & Health, Center for Food Innovation, Delft 2613 AX, The Netherlands

https://orcid.org/0009-0002-3946-4067
van Peij Noël N M E dsm-firmenich, Taste, Texture & Health, Center for Food Innovation, Delft 2613 AX, The Netherlands

https://orcid.org/0000-0001-5502-4729
Cambillau Christian School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland
Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IMM), Aix-Marseille Université – CNRS, UMR 7255 Marseille, France

https://orcid.org/0000-0001-5846-6303
Mahony Jennifer School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

https://orcid.org/0000-0003-1823-7957
van Sinderen Douwe School of Microbiology & APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

To whom correspondence should be addressed. Tel: +353 214901365; Email: d.vansinderen@ucc.ie
The first two authors should be regarded as Joint First Authors.

09 9 2024
09 8 2024
09 8 2024
52 16 97609776
22 7 2024
17 7 2024
17 5 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
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Abstract

Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called ‘defence islands’ in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems.

Graphical Abstract

Graphical Abstract

Science Foundation Ireland 10.13039/501100001602 firmenich 10.13039/100018220 12/RC/2273‐P1 12/RC/2273‐P2 17/SP/4678 GENCI 10.13039/501100010190 2023-AD010714075 National Institutes of Health 10.13039/100000002 R01-GM129325 National Institute of Allergy and Infectious Diseases 10.13039/100000060
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pmcIntroduction

Lactococcus lactis and Lactococcus cremoris (formerly assigned as two subspecies of Lactococcus lactis) (1) are bacterial species of substantial economic and industrial importance due to their extensive use in the production of fermented food products, such as cheese, buttermilk and sour cream (2–4). These lactococcal species are characterized as Gram-positive, non-spore forming, micro-aerophilic coccoid bacteria that belong to the lactic acid bacteria (LAB) (5).

The widespread and intensive application of lactococcal strains is associated with the emergence of host-specific bacteriophages, which are ubiquitous in the dairy environment and which represent a persistent challenge to fermentation processes (6). Phage infection may cause (partial) elimination of the starter culture, resulting in delayed or even failed fermentations with severe economic consequences for producers (7). Among the described lactococcal phage groups/genera (8,9), members of the Skunavirus genus (formerly 936 phage group), Ceduovirus genus (formerly c2 phage group) and P335 group are particularly prevalent and problematic in modern, large-scale dairy fermentation plants (10).

To defend themselves against phages, bacteria have adopted multiple phage resistance strategies that interfere with different stages of the phage infection cycle, such as preventing phage adsorption or phage DNA injection, restriction of incoming phage nucleic acids (Restriction-Modification [RM] and Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein [CRISPR-Cas] systems) and abortive infection (Abi) (11,12). CRISPR-Cas systems are typically not present in Lactococcus with very limited reports of their presence in recent studies (13,14). Abi systems instead represent one of the most common lactococcal antiphage mechanisms. They are activated upon phage infection preventing phage proliferation and subsequent infection of neighbouring cells (12,15). To date, 23 genetically distinct and mechanistically diverse lactococcal Abi systems have been reported, designated AbiA to AbiZ (12,16). The majority of the currently defined lactococcal Abi systems are plasmid-encoded, typically by one or two genes, and with diverse modes of action interfering with critical stages of the phage lytic cycle, including DNA replication, transcription and protein production (5,16).

Despite intense research efforts focused on antiphage systems in lactococci until the 2000s (12), the recent discovery of many previously unknown antiphage systems using computational and high-throughput methods in other bacteria demonstrates that the phage resistance landscape is far more extensive and varied than previously imagined. More specifically, a multitude of studies have reported on the exploration of genomic data sets for the identification of novel antiphage systems in primarily Escherichia coli and Bacillus subtilis as model and test organisms (17–22). Furthermore, most of the recent studies have mined whole genome sequences and are largely based on the genomic co-localization with known antiphage systems (so-called ‘defence islands’) for high-throughput bioinformatic prediction and discovery of antiphage systems (17–20). However, a recent experimental screening approach based on random cloning of genome fragments has also allowed discovery of antiphage systems, indicating their ubiquitous nature (21).

Although it has recently been suggested that mobile genetic elements (MGEs) may frequently carry antiphage systems and represent major contributors to their horizontal transfer (20–24), plasmids have not thus far been systematically searched for antiphage systems. Dairy-associated lactococcal strains typically harbour several plasmids, with a report of an isolate containing as many as twelve (25). In a large-scale analysis of lactococcal plasmids, an average of five plasmids per strain was identified, ranging in size from 0.9 kb to 193 kb and accounting for up to 355 kb of extra-chromosomal DNA (14), which is a remarkable feature considering that the genome size of individual lactococcal strains can range between 2242 and 2688 kb (26). Many biotechnologically relevant traits, such as casein degradation, lactose utilization and citrate metabolism are plasmid-encoded (14,16). However, the function of most lactococcal plasmid-associated gene products remains unknown and it may well be that some of these confer antiphage activity.

The aim of the current study was to survey the combined plasmidome of 53 lactococcal strains for encoded antiphage systems and to provide insights into the structure and function of the identified systems, thus expanding our understanding of the bacterial immunity landscape and generating a phage resistance-encoding plasmid library that can be applied in the dairy industry for improved strain robustness or beyond.

Materials and methods

Strains, media and growth conditions

Bacterial strains, plasmids and bacteriophages used in this study are listed in Supplementary Table S1. Lactococcal strains used in this study were grown at 30°C in M17 broth (Oxoid) supplemented with 0.5% (w/v) glucose (GM17) for 16–20 h. GM17 was supplemented with either chloramphenicol (5 μg/ml, Sigma-Aldrich, to select for strains carrying pNZ44 or its derivatives), tetracycline (5 μg/ml, Sigma-Aldrich, to select for strains harbouring pPTPi or its derivatives) or erythromycin (10 μg/ml, Sigma-Aldrich, for pPEPi- or derivative-containing strains). Nisin from L. lactis (1–10 ng/ml, Sigma-Aldrich) was added to growing cultures when an OD600nm of 0.2–0.3 was reached for the induction of the PnisA promoter of pPTPi-derivative plasmids. Where appropriate, GM17 agar plates were also supplemented with nisin as described above.

Escherichia coli strains were grown overnight at 37°C with agitation (150 rpm, Multitron Standard, Infors HT) in LB broth (Thermo Fisher Scientific) supplemented with 10 μg/ml tetracycline, to select for strains harbouring pPTPi or its derivatives. For the preparation of GM17 or LB agar plates 1.5% (w/v) bacteriological agar was added.

Bacteriophage propagation

Bacteriophages used in this study were propagated by adding 2–5% (v/v) of a fresh overnight culture of the appropriate lactococcal host strain, CaCl2 (10 mM) and 1% (v/v) of the phage lysate (108–1010 pfu/ml) or a single plaque in GM17 broth. Incubation was continued at room temperature or 30°C until lysis occurred. The lysates were filtered (pore size 0.45 μm, Sarstedt AG & Co. KG) and stored at 4°C. The phages used in this study belong to one of four genera of lactococcal phages which have previously been shown to be genetically distinct (9). The Skunavirus members employed in this study share over 90% nucleotide sequence similarity over at least 70% of their genomes highlighting the intra-genus diversity of these phages based on BLASTN alignments, while the P335 phages used have previously been shown to be highly genetically diverse (27).

DNA extraction, sequencing, assembly and annotation of lactococcal strains

Genomic DNA of 53 lactococcal strains was isolated from a fresh 10 ml overnight culture of each strain using a NucleoBond® DNA extraction kit with Buffer set III (Macherey-Nagel) according to the manufacturer's instructions, with the exception of the addition of mutanolysin (Sigma-Aldrich; 50 U/ml) to Buffer G3 prior to an extended (16–18 h) incubation at 37°C. DNA was stored at –20°C prior to shipment to the sequencing facility.

Genome sequencing was performed by a combined SMRT (long read) and Illumina (short read) approach on a Pacific Biosciences RS II sequencing platform (executed by Eurofins Genomics, Germany) and an Illumina MiSeq platform (executed by GenProbio s.r.l., Italy), respectively. De novo hybrid assemblies using both datasets were performed using the Unicycler v0.5.0 hybrid assembly pipeline (28). Open Reading Frame (ORF) prediction was performed with Prodigal v2.5 prediction software (29) and confirmed using BLASTX v2.2.26 alignments (30). ORFs were automatically annotated using BLASTP v2.2.26 (30) analysis against the non-redundant protein databases curated by the National Centre for Biotechnology Information (NCBI). Artemis v18 genome browser (31) and annotation tool was used to manually curate ORFs and for the combination and inspection of ORF results. Final ORF annotations were refined where necessary using Pfam (32) and/or HHpred (33). Extrachromosomal contigs were annotated (as above) and manually curated (using BLAST) to confirm the presence of one or more previously described lactococcal plasmid-borne rep genes. Contigs of appropriate sizes containing at least one rep gene were determined to be plasmids.

Extracting gene families from plasmid sequences

All-against-all, bi-directional BLAST alignments were performed for all sequence comparisons at protein level. An alignment cut-off value of > 50% amino acid identity across 50% of the sequence length was used (with an associated E-value of <0.0001). The Markov Clustering Algorithm (MCL) was applied for analysis and clustering of these results in the mclblastline pipeline v12-0678. TM4 MeV, MultiExperiment Viewer v4.9 was employed to view MCL clustering data and to conduct hierarchical clustering (HCL) (https://webmev.tm4.org/about) (Supplementary Table S2).

Selection criteria and analysis of the candidate systems

At least one representative gene sequence of each family (Supplementary Table S2) was subjected to comparative sequence analysis (BLASTN and BLASTP) (30) against available sequence data on the NCBI database (34) and to protein homology prediction analysis using HHpred (33) and InterPro (35). TMHMM v.2.0 software was used for the prediction of transmembrane regions in these proteins using the hidden Markov model (HMM) (36). The selection criteria for putative antiphage systems were rather loose and based on properties of previously identified lactococcal Abi systems (12). Specifically, single or pairs of genes with a high AT% content (preferably ≥ 70%) and encoding proteins, predominantly of unknown function and preferably with no transmembrane domains, were manually selected. Additionally, all plasmid sequences were submitted into DefenseFinder v1.0.9 (37,38) and PADLOC v1.1.0 (39) (databases v1.2.1 and v1.4.0, respectively) to search for established antiphage systems.

Cloning of candidate antiphage systems

DNA fragments to be cloned were amplified using Phusion Green High-Fidelity DNA Polymerase (Thermo Fisher Scientific), Phusion High-Fidelity DNA Polymerase, Q5 High-Fidelity DNA Polymerase or OneTaq DNA Polymerase (all New England Biolabs) according to the manufacturer's instructions and with relevant primers (Supplementary Table S2; Eurofins Genomics). The initial denaturation step was performed for 3–10 min to allow cell disruption and release of the template DNA. The high-copy, constitutive-expression lactococcal plasmid pNZ44 was used as a cloning vector (Supplementary Table S2), although in some cases DNA fragments, which appeared to be recalcitrant to cloning in pNZ44, were instead cloned in the low-copy, nisin-inducible vector, pPTPi (Supplementary Table S2).

Nine candidates that could not be amplified by PCR were ordered as synthetic genes from Eurofins Genomics. Candidate genes were excised using restriction digestion from synthetic constructs and restricted DNA fragments were manipulated as described below.

FastDigest restriction enzymes, including PstI, KpnI, XbaI, HindIII, SalI, SacI, EcoRI, BamHI (Supplementary Table S2; Thermo Fisher Scientific) and T4 DNA ligase (Promega) were used according to the manufacturer's instructions. Restricted DNA fragments and corresponding cloning vector backbones were purified prior to ligation using the GenElute PCR Clean-up Kit (Sigma-Aldrich). Ligation mixtures were introduced into L. lactis NZ9000 by electroporation (see next paragraph) and transformants were selected based on either chloramphenicol, tetracycline or erythromycin (for the presence of [derivatives of] pNZ44, pPTPi or pPEPi respectively). Two candidates (Supplementary Table S2, no. 129 and 130) were cloned by circular polymerase extension cloning (CPEC, Quan and Tian, 2011), using the primers indicated at the end of Supplementary Table S2 to prepare the linearized vectors and E. coli EC101 as the host.

Transformants carrying the desired recombinant plasmids were screened by colony PCR using vector-specific primers (pNZ44_F and pNZ44_R or pPTPi_F and pPTPi_R, Supplementary Table S2). Plasmid DNA was extracted from the positive clones using the GeneJET Plasmid Miniprep/Maxiprep Kit (Thermo Scientific) according to the manufacturer's instructions with the following modifications. Harvested cells were resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) containing 25% sucrose and 30 mg/ml lysozyme (Sigma Aldrich) and incubated for 30 min at 37°C prior to the plasmid DNA extraction procedure to facilitate the degradation of the cell wall. Sequence integrity of the recombinant plasmids was verified by Sanger sequencing (Eurofins Genomics).

Lactococcal electrocompetent cells were prepared by inoculating 40 ml of GM17 supplemented with 0.5 M sucrose and 1% (for L. cremoris 3107) or 1.5% glycine (for L. cremoris NZ9000 and L. lactis IL1403) with up to 5% of fresh or pre-adapted overnight culture. The culture was incubated at 30°C until an OD600nm of 0.5 was reached. Cells were harvested, washed, aliquoted and stored as previously described. 45 μl of competent cell suspension was mixed with 5 μl of the ligation mixture and electroporated at 2.0 kV, 200 Ω and 25 μF in 0.2 cm cuvettes using an ECM 630 Exponential Decay Wave Electroporation System (BTX). Following electroporation, cells were recovered in 950 μl GM17 broth containing 20 mM MgCl2 and 2 mM CaCl2 and incubated at 30°C for 2.5 h. Bacteria were then plated on GM17 supplemented with relevant antibiotics for selection of pNZ44 or pPTPi and incubated at 30°C for 24–48 h.

Escherichia coli EC101 electrocompetent cells were prepared by inoculating 500 ml of LB broth with 5 ml of overnight culture. The culture was incubated at 37°C with shaking until an OD600nm of 0.8 was reached. Cells were harvested at 4500 rpm at 4°C for 10 min and were then washed twice with 100 ml of ice-cold ELGA water and once with 50 ml of 10% ice-cold glycerol. The pellet was resuspended in 2.5 ml of 10% glycerol and aliquots of 100 μl were stored at –80°C. E. coli competent cells were electroporated as described for the lactococcal cells except using a voltage of 2.5 kV and were recovered in LB broth with no supplementations. Bacteria were plated on LB agar supplemented with 10 μg/ml tetracycline for the selection of pPTPi derivatives and incubated at 37°C for 24 h.

Spot and plaque assays

Spot and plaque assays were performed using the double agar method of Lillehaug (40) with some modifications. Solid (bottom layer) and semi-solid agar (top layer) were prepared using GM17 medium supplemented with CaCl2 (10 mM); 1% bacteriological agar was incorporated in the solid layer, and 0.4% agar was used in the semi-solid agar. To enlarge the plaque size, semi-solid agar was supplemented with 0.2% agarose instead of agar or 0.4% glycine was added to both layers where required. SM buffer (10 mM CaCl2, 100 mM NaCl, 10 mM MgSO4, 50 mM Tris–HCl at pH 7.5) was used as the diluent in all bacteriophage assays. The efficiency of plaquing (EOP) was determined by dividing the titre (PFUs/ml) of the test strain by that of the control strain. An antiphage system was considered to contribute to plaque size reduction if the plaque size range of the phage plated on the strain expressing the antiphage system was substantially smaller compared to the plaque size range of the same phage plated on the wild type strain under the same conditions. An example of plaque counts, EOP calculation with standard deviation and plaque size range measurements can be found in Supplementary Table S3.

Adsorption and transduction assays

Adsorption assays were carried out following the protocol described by Mosterd and Moineau (41) with some modifications. Specifically, host cells were grown at 30°C and CaCl2 was added to a final concentration of 10 mM.

Transduction assays were based on the protocol of McGrath et al. (42). To prepare sk1 and c2 transduction lysates, phages sk1 and c2 were propagated on L. cremoris MG1363 harbouring pPTPL-sk1cos or on L. cremoris NZ9000 carrying pPTPi-c2cos, respectively (Supplementary Table S2). At an OD600nm of 0.2, strains to be tested were infected with transduction lysate at a multiplicity of infection (MOI) of 1. CaCl2 was added to a final concentration of 10 mM and after 10 min of incubation at 30°C cultures were plated on solid growth medium supplemented with 5 μg/ml tetracycline for the selection of the transductants. Transduction frequencies were calculated as number of transductants divided by the total number of phage particles applied.

Lysis-in-broth assay

Overnight cultures of L. cremoris NZ9000 carrying pNZ44/pPTPi and their antiphage system derivatives were subcultured (2%) in GM17 broth medium and incubated at 30°C until an OD600nm of 0.2 was reached. CaCl2 was added to a final concentration of 10 mM and an equal volume of phage sk1 or c2 was added to a final MOI of 0.05 and 5. For the uninfected control culture, an equal volume of spun-down and filtered overnight culture was used. 200 μl of each culture was then transferred into a 96 wells plate. Plates were incubated at 30°C and OD600nm measurements were recorded every 30 min for a total of 300 min with a MULTISKAN FC plate reader (Thermo Fischer Scientific).

Alphafold2 structure and domain predictions

We performed predictions with either a Colab notebook running AlphaFold v2.3.1 (43,44) (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb) or HPC resources from GENCI-IDRIS running AlphaFold v2.3.1 (45). The pLDDT values (Supplementary Figure S5) of predicted structures were stored in the PDB files as B-factors and plotted with the Colab or the IDRIS resources. The final predicted protein or domain structures were submitted to the Dali server (46) to identify the closest structural homologues in the PDB. Visual representations of the structures were prepared with ChimeraX (47).

Assessing prevalence by BLASTP and HMMER searches

Antiphage systems were subjected to NCBI’s non-redundant protein sequence database (BLASTP (48)). A cut-off value of >70% amino acid identity with an associated E-value of <0.001 was used to factor the distinct sequence hits or organisms. Similarly, the new antiphage sequences were subjected to HMMER (49). NCBI’s non-redundant protein sequence database (34) filtered for a maximum pairwise sequence identity of 70% was selected with a cut-off E-value of <0.001 for the target sequences, while a maximum number of 10 000 hits was selected to be displayed.

Results

Selection of candidate plasmid-encoded antiphage systems

To identify previously unknown, non-RM antiphage systems encoded by lactococcal plasmids, a total of 321 plasmid sequences derived from 53 sequenced lactococcal strains in our collection (University College Cork and dsm-firmenich) were analysed. These strains were found to harbour four to 10 plasmids, with an average of six plasmids per strain. Based on HCL analysis, the 819 589 identified plasmid-borne coding sequences were grouped into 1019 distinct genes families (Supplementary Table S2). For a gene sequence to be selected as a candidate antiphage system, relaxed criteria were applied based on the generic properties of previously identified lactococcal Abi systems (see methods section).

Genes that encode proteins identical (AbiA, AbiE, AbiF, AbiG, AbiJ, AbiP and AbiZ) or similar (11 AbiB-like, AbiC-like, AbiD-like and AbiR-like; ranging from 38.4 to 99.8% amino acid identity) to the 23 previously established lactococcal Abi systems (12) were also identified during the candidate gene selection and were included in our experimental approach to validate our identification methodology and to provide a more complete overview of all non-RM antiphage systems present in our surveyed plasmidome. In addition to these 11 systems, a homologue encoding the AbiQ toxin (50) was present in our plasmid collection, though apparently in the absence of its associated antitoxin and was therefore not included in our experimental approach. Interestingly, for 11 out of the 23 currently known lactococcal Abi systems, no homologues were found within the 1020 analysed gene families representing our plasmid collection.

To identify additional antiphage systems not previously confirmed or described in Lactococcus, the lactococcal plasmidome was also subjected to bioinformatic scrutiny using PADLOC (Prokaryotic Antiphage Defence LOCator) and DefenseFinder (37–39). This resulted in the prediction of eight antiphage systems previously shown to be active in bacteria other than Lactococcus lactis/cremoris (i.e. Bunzi, Cyclic oligonucleotide-based antiphage signaling system [type I and II CBASS], DUF2290/Helicase, Lamassu, phage anti-restriction-induced system [PARIS], RnlAB and Septu type I).

In total, we selected 198 candidate antiphage-encoding systems (153 of which represent a single gene, while the remaining 45 consist of more than one gene), together representing 232 different gene families (Supplementary Table S2).

Identification of plasmid-encoded antiphage systems

The 198 candidate antiphage systems were cloned in pNZ44 (high copy vector) or pPTPi (low copy vector) and introduced into L. cremoris NZ9000. Following sequence verification of the resulting recombinant plasmids, plaque assays were performed against a selection of lactococcal phages to determine if the presence of a given recombinant plasmid was associated with phage resistance.

Of these 198 tested candidates, 20 were shown to provide resistance against the tested phages (discussed in detail below). Eight of these were homologues of the previously established lactococcal Abi systems (AbiA, AbiB-like, AbiD-like, AbiF, AbiG, AbiJ, AbiP and AbiZ with AbiB-like and AbiD-like exhibiting 94% and 59% amino acid sequence identity to AbiB and AbiD, respectively). Interestingly, AbiF-mediated phage resistance activity was initially not observed when abiF was cloned with the native or an artificial Shine Dalgarno sequence derived from pNZ8048 (Supplementary Table S2). However, homology at amino acid sequence level had previously been reported between AbiD, AbiD1 and AbiF (51,52,53). For AbiD1, it had been shown that a short upstream sequence containing a strong promoter and terminator was required for its antiphage activity (52). When analysing if abiF and abiD-like are preceded by a similar sequence structure, the sequences upstream of abiF and abiD-like were found to be 80% and 68% identical, to that upstream of abiD1 and abiD respectively. Cloning abiF and abiD-like with their respective upstream promoter and terminator sequences resulted in constructs which upon testing were shown to exhibit antiphage activity in the particular set-up as assessed here. Interestingly, several systems similar to established Abi systems showed no antiphage activity. In more detail, only one out of the eleven AbiB-like constructs (94% amino acid identity to AbiB) was shown to elicit anti-phage activity. The remaining (ten) assessed and apparently inactive AbiB-like systems shared 92–99% identity at amino acid level though with varying coverage which in many cases appeared to be due to gene truncations. AbiC-like shares just 38% amino acid sequence identity to the previously described AbiC and provided no resistance against the tested phages. The absence of AbiE activity is probably due to its phage specificity, as it had previously been shown to be active only against one particular phage (out of several tested) (54), of which we did not have an exact copy in our collection. AbiR-like was not analysed due to cloning issues.

Among the eight systems identified by PADLOC and DefenseFinder (i.e. Bunzi, type I and II CBASS, DUF2290/Helicase, Lamassu, PARIS, RnlAB and Septu, Supplementary Table S2), antiphage activity of five lactococcal homologues (PARIS, type I and II CBASS, Lamassu and Septu) was confirmed in our Lactococcus test set-up. Bunzi, DUF2290/Helicase and RnlAB did not offer any protection activity against tested lactococcal phages.

Based on comparative sequence analysis (BLASTN and BLASTP) and on functional and structural analysis (HHpred and InterPro) the remaining seven antiphage systems (out of the twenty identified) did not display significant homology to any previously described antiphage system, thus rendering them novel antiphage systems (Table 1). These systems were named after cheese, cattle or fermentation related deities (Rhea, Aristaios, Kamadhenu, Fliodhais, Audmula, Rugutis and Hesat), following a similar naming approach as established by Doron et al. (17). Except for Hesat, which was cloned in pPTPi with the addition of its native signals, all novel systems were cloned in the high-copy number vector pNZ44 (Supplementary Table S2). All discovered systems, except Fliodhais, are single gene antiphage systems. Both genetic components are necessary for Fliodhais activity; the described antiphage activity (see below) was not observed when the two components were cloned and tested separately.

Table 1. Names, genes loci and predicted domains of the discovered antiphage systems

	System name	Gene locus	Protein size (aa)	InterPro Prediction	HHpred Prediction	AlphaFold2/Dali Prediction	Associated InterPro/Pfam	Associated HHpred	
New antiphage systems	Rhea	pUCCL617_141	303	-	-	transposase TnsE (62)	-	-	
	Aristaios	pUCCL641_199	316	DUF2971	-	ADP-ribosyltransferase (64)	IPR021352/PF11185	-	
	Kamadhenu	pUCCL624_130	180	YfbU (66)	YfbU	YfbU	IPR005587/PF03887	1WPB_F	
	Fliodhais	pUCCL632_186	41	-	lipoprotein	membrane protein	-	5CYB_A	
		pUCCL632_185	255	-	-		-	-	
	Audmula	pUCCL630_365	392	PBP transglycosylase	PBP	transglycosylase	IPR036950	2JCH_A	
	Rugutis	pUCCL633_059	359	SEC-C motif	SEC-C motif	SEC-C motif	IPR004027/PF02810	2I9W_A	
	Hesat	pUCCL620_156	213	-	-	colicin-D toxic domain (68)	-	-	
Systems new in Lactococcus/ -like systems	PARIS	pUCCL620_250	453	AAA_21 ATPase (20)	SMC protein	Rad50 ATPase	IPR003959/PF13304	6QJ0_A	
		pUCCL620_249	280	DUF4435 (20)	Hypothetical protein PH0156	TOPRIM domain of OLD nucleases (20)	IPR029492/PF14491	2P62_A	
	Type I CBASS	pUCCL631_167	190	SLATT effector (72)	-	SLATT effector	IPR040811/PF18186	-	
		pUCCL631_168	408	NTase/AGS-C sensor (70)	CD-NTase (72)	CD-NTase	IPR006116/IPR040511/PF18134	7LJO_A	
	Type II CBASS	pUCCL620_247	286	SUa-2TM effector (72)	-	Cap5 SAVED (71)	IPR041502/PF18179	-	
		pUCCL620_246	383	NTase/SMODS (72)	CD-NTase	CD-NTase	IPR006116/PF18144	7LJN_C	
		pUCCL620_245	155	-	Ubiquitin-conjugating enzyme E2a	Cap2 (72)	-	7JZV_A	
	Lamassu	pUCCL630_136	427	ABC-3C system CTD	Cap4 endonucleasea (19)	Cap4 SAVED/CARF domain	IPR046920/PF20276	7YIB_A	
		pUCCL630_135	242	ABC-3C system MC	-	CDI DNAse	IPR046905/PF20289	-	
		pUCCL630_134	883	AAA_23 Rad50/SbcC ATPase	SMC protein (19)	Rad50 ATPase	IPR038729/PF13476	7TVE_E	
	Septu	pUCCL618_274	537	AAA_21 ATPase (17)	ARE-ABCF protein	excinuclease ABC	IPR003959/PF13304	8BUU_9	
		pUCCL618_273	443	HNH endonuclease (17)	Cas9 endonuclease	HNH endonuclease/GAF domain	IPR003615/PF01844	7ENH_A	
	AbiD-like	LLA22_pB0040	345	AbiD/AbiF-like	-	Cas13d ribonuclease (54)	IPR017034/PF07751	-	
The associated HHpred represents the highest probability hit (> 97%) with an E-value below 0.001 except for a (probability > 95%, 0.01 ≤ E-value ≤ 0.05). DUF (domain of unknown function); PBP (penicillin binding protein); AAA (ATPases associated with diverse cellular activities); SMC (structural maintenance of the chromosome); TOPRIM, (topoisomerase-primase); SLATT (SMODS [second messenger oligonucleotide or dinucleotide synthetases] and LOG-associating two TM [transmembrane]); AGS-C, (Adenylyl, Guanylyl & SMODS sensor C-terminal); CD-NTase (cGAS/DncV-like nucleotidyltransferase; Sua-2TM (SMODS/Ubiquitin system-associated 2TM); Cap (CD-NTase-associated protein); SAVED (SMODS-associated and fused to various effector domains); ABC (ATP-binding cassette); CTD (C-terminal domains); CARF (CRISPR-associated Rossman fold); MC (Middle Component); CDI (contact-dependent growth inhibition); ARE-ABCF (antibiotic resistance ABC protein of the F subfamily); Cas (CRISPR-associated); GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA)

Antiphage spectrum of the verified antiphage systems

The antiphage activity spectrum of the twenty antiphage systems when present in L. cremoris NZ9000, L. cremoris 3107 or L. lactis IL1403 was evaluated by challenging these strains with various lactococcal bacteriophages (representing the Skunavirus, Ceduovirus, P335 group and Teubervirus genera).

Figure 1 (Detailed information is presented in Supplementary Table S3) summarizes the efficiency of plaquing (EOP) of each tested phage on the lactococcal strain harbouring a given antiphage system compared to the corresponding wild type strain carrying the empty vector (pNZ44 or pPTPi). In this experimental setup, all 20 systems, except Fliodhais and Audmula, provided (varying levels of) resistance against at least one member of the Skunavirus genus, which are the most frequently encountered phages in the dairy industry (55). Furthermore, the majority of the systems was shown to exhibit a similarly high level of resistance (over four log EOP reduction) against all assessed skunaviruses. Varying levels of resistance across assessed skunaviruses were observed for Rugutis, AbiG, AbiJ and AbiF. The anti-Skunavirus activity of Rugutis, AbiG and AbiJ is strain-dependent, whereas the activity of AbiF was shown to be phage-specific in our tests. Eleven out of the 18 systems that provide resistance against skunaviruses also cause noticeable plaque size reduction (Figure 1).

Figure 1. Efficiency of plaquing (EOP) of different phage genus representatives (Skunavirus, Ceduovirus, P335 group & Teubervirus) on different lactococcal host strains (NZ9000, 3107, IL1403) carrying an antiphage system. Detailed EOP information summarized in this figure is presented in Supplementary Table S3. Data represent the mean of three biological replicates. As the nisin-inducible PnisA promoter requires the NisRK two-component system, which is not present in L. cremoris 3107 or L. lactis IL1403, the pPTPi-derived constructs were only tested in L. cremoris NZ9000. Additionally, some of the generated expression constructs, although introduced and tested in L. cremoris NZ9000, could not be introduced or were not stable in L. cremoris 3107 or L. lactis IL1403 (Rugutis, type I and II CBASS, AbiG, AbiJ and AbiZ), and therefore the corresponding EOP values could not be defined.

Ten systems were shown to provide varying levels of resistance against the Ceduovirus c2, another dominant lactococcal phage genus (56). Fliodhais was associated with smaller c2 plaques surrounded by a halo (<1 mm clear-centred plaques with a faint ring), a phenotype that was not observed when phage c2 was plated on the wild type strain (up to 4 mm clear-centred plaques without a halo). Resistance against the P335 phage group was limited to five systems and a single phage (P335), except for Rhea which was unique in providing high resistance against phage TP901-1. It should be noted however that many systems were not tested against this phage group, as many strains could not be constructed due to various issues (Figure 1). None of the tested systems was effective against the Teubervirus P087.

The newly identified lactococcal antiphage systems are active post phage DNA injection

To examine if the newly identified antiphage systems are active extracellularly or intracellularly, before or after phage adsorption/DNA injection, phage adsorption and transduction assays were performed. Fliodhais, whose antiphage effect was limited to plaque size reduction, and previously characterized lactococcal Abi systems were excluded from these characterization assays.

Adsorption assays (Figure 2A-C) revealed that the assessed systems do not interfere with phage sk1 or c2 adsorption. Similarly, DNA injection does not appear to be noticeably affected by the tested antiphage systems as the observed transduction frequencies are comparable to those obtained for the control strains (Figure 2D–F). Transduction frequencies observed in the presence of the Audmula and PARIS systems were slightly higher (from [1.69 ± 0.37] × 10−4 to [2.71 ± 0.23] × 10−4 and from [2.90 ± 0.44] × 10−5 to [3.70 ± 0.71] × 10−5 respectively) than the control strains. Conversely, Rhea, Aristaios and Rugutis antiphage systems were associated with an observed transduction frequency reduction of up to one log. A possible explanation for these differences can be the overexpression of the systems interfering with other cellular activities and thus slightly with phage DNA injection. However, this effect is negligible when compared to the observed transduction frequency reduction of a strain expressing the known DNA injection blocking protein Sie2009 (57), whose transduction frequencies were below the detection limit (<1.05 × 10−7). Therefore, these antiphage systems do not appear to specifically target phage adsorption or DNA injection steps, indicating that they are active intracellularly at a stage beyond phage DNA injection.

Figure 2. Adsorption and transduction assays. Adsorption percentages of phages sk1 (panel A) and c2 (panel B) to L. cremoris NZ9000 strains carrying antiphage systems compared to control strains L. cremoris NZ9000::pNZ44 and L. cremoris NZ9000::pPTPi induced at 1 ng/ml (panel C I) or 10 ng/ml nisin (panel C II). Transduction frequencies for L. cremoris NZ9000::pNZ44 derivatives transduced with pPTPL-sk1cos (panel D) or pPTPi-c2cos (panel E) or L. cremoris NZ9000::pPEPi derivatives induced at 1 ng/ml (panel F I) or 10 ng/ml (panel F II) transduced with pPTPL-sk1cos. Experiments were performed in biological triplicate and data are presented as means ± standard deviation (SD). Asterisks mark statistically significant differences in transduction frequencies between the samples and the relevant controls (unpaired t-test, P-value < 0.05).

Most of the newly identified antiphage systems present Abi phenotypes

To assess if the newly identified antiphage systems exhibit phenotypic characteristics typical of abortive infection systems, lysis profiles of L. cremoris NZ9000 and its derivative strains expressing antiphage systems were determined at a low (0.05) and a high (5) multiplicity of infection (MOI) of phage sk1 or c2 (Figure 3). A phenotype of providing phage resistance at a low MOI, yet entering a bactericidal/bacteriostatic state at a high MOI is consistent with an antiphage system acting through abortive infection (15,58). All tested systems provided phage resistance under low MOI conditions, growing at rates comparable to those of uninfected strains, while corresponding cultures carrying the empty vectors were shown to collapse. The exception to this was Audmula, where delayed lysis occurred 120 min later than the phage-sensitive control, L. cremoris NZ9000::pNZ44. However, at a high MOI, all cultures, including those expressing the newly identified antiphage systems demised or entered a state of bacteriostasis. Therefore, all assessed systems, except Audmula, phenotypically behave like an Abi system, though further experimentation is required to validate this (59). It should be noted that the uninfected control for several strains expressing antiphage systems (notably PARIS and Lamassu) exhibited a decreased growth rate, indicating that expression of certain systems comes at a fitness cost, consistent with previous observations (60,61).

Figure 3. Lysis-in-broth assays. Lysis-in-broth graphs for L. cremoris NZ9000::pNZ44 and various antiphage system-expressing derivatives infected by phages sk1 and c2 (panel A), as well as for L. cremoris::pPTPi and various antiphage-expressing derivatives infected by phage sk1 (panel B). Each growth curve represents the mean of three biological replicates and the shaded area corresponds to the standard deviation.

Domain and structure prediction of the novel antiphage systems

To obtain information on the possible mode of action for each of the identified antiphage systems, the individual protein(s) of each system were structurally analysed with AlphaFold2 and Dali, and the obtained results were then compared with functional domain predictions obtained by HHpred and InterPro (Table 1). The associated HHpred prediction represents the highest probability hit (> 97%) with an E-value below 0.001. The Alphafold2 pLDDT values and predicted aligned errors (PAE) plots are present in Supplementary Figure S5. Z-scores and rmsd values are presented in the figure legends.

Employing HHpred and InterPro, no significant protein domain predictions were found for Rhea. However, based on AlphaFold2 analysis, the C-terminus of Rhea resembles the DNA-binding protein TnsE (Protein Data Bank [PDB] ID 5D17; Figure 4A, B), which is part of a multicomponent transposon complex (62). TnsE is believed to recognize replication intermediates that are typical of conjugative plasmids or of replicating phages, suggesting that this domain is responsible for sensing phage infection with subsequent activation of the antiphage system. Such a mechanism would be reminiscent of type II Lamassu, which protects Vibrio cholerae from both phage infection and plasmid invasion by recognizing (phage) DNA replication intermediates (63).

Figure 4. Predicted structures of novel antiphage systems. (A) Ribbon structure of Rhea (rainbow coloured). (B) Superimposition of Rhea (blue) with PDB 5D17 (grey), a transposase element (Z = 6.2, rmsd = 3.2 Å). (C) Ribbon structure of Aristaios (rainbow coloured). (D) Superimposition of Aristaios (blue) with PDB 5ZJ5 (grey), an ADP-ribosyltransferase (Z = 5.6, rmsd = 3.6 Å). (E) Aristaios molecular surface (blue) with ADP from 5ZJ5 in the catalytic crevice. (F) Ribbon structure of the two-domain protein Kamadhenu (rainbow coloured; no significant hit in the PDB). (G) Superimposition of Kamadhenu (blue) with PDB 1WPB (grey), the YfbU protein forming a large complex of unknown function (Z = 16.4, rmsd = 2.2 Å). (H) Ribbon structure of Audmula (rainbow coloured; the 5 trans-membrane helices are boxed in grey). (I) Superimposition of Audmula (blue) with PDB 3VMR (grey), a transglycosylase; (Z = 9.8, rmsd = 6.4 Å. 3VMR possesses a unique transmembrane helix). (J) Audmula molecular surface (blue) with Lipid II from 3VMR in the catalytic crevice. (K) Ribbon structure of Fliodhais-ii (rainbow coloured). Several hits with membrane proteins (boxed in grey) do not point to a defined function. Fliodhais-i (not presented here) was not reliably predicted by AF2, including Fliodhais-i/ii complexes. (L) Fliodhais-ii molecular surface (coloured according to hydrophobicity, brown). Residues 10–32 form a trans-membrane helix. (M) Ribbon structure of Rugutis (rainbow coloured; no significant hit in the PDB). (N) Ribbon structure of Hesat (rainbow coloured). (O) Superimposition of Hesat (blue) with PDB 1V74 (grey), a colicin-D toxic domain (Z = 4.9, rmsd = 2.7 Å). pLDDT values and predicted aligned errors (PAE) plots present in Supplementary Figure S5.

The Aristaios antiphage system contains a domain of unknown function (DUF2971) based on HHpred and InterPro searches. AlphaFold2 analysis revealed that an ADP-ribosyltransferase domain covers most of Aristaios (PDB ID 5ZJ5; Figure 4C–E). Such a protein fold is present in a family of bacterial toxins (ADP-ribosylating toxins) that inhibit essential host protein (and potentially phage) functions (64), likely underpinning the abortive infection phenotype elicited by Aristaios. Recently, a DarTG toxin-antitoxin system has been described that depends on the DNA ADP-ribosyltransferase activity of its DarT toxin to modify invading phage DNA and prevent phage replication (65).

AlphaFold2 revealed that Kamadhenu contains a YfbU (PDB ID 1WPB; Figure 4F, G), a two-domain helical protein, which has been reported to be involved in cell death when triggered by DNA damage (66). This would therefore indicate a mechanistic phenomenon in accordance with the observed abortive infection phenotype of Kamadhenu.

The N-terminus of Audmula contains four transmembrane helices, followed by a large C-terminal helical domain that resembles a transglycosylase (PDB ID 3VMR; Figure 4H, I). This suggests that Audmula, the expression of which results in delayed lysis, is involved in cell wall modification, possibly affecting the lysis stage of the phage infection cycle. Cell wall modification resulting in delayed lysis of phage infected cells has previously been demonstrated in B. subtilis, where dynamin-like protein DynA forms complexes, thereby possibly stabilizing the cell membrane (67).

Based on HHpred outputs, the first component of Fliodhais resembles a lipoprotein that is associated with virulence in Streptococcus pneumoniae. AlphaFold2 prediction reveals that Fliodhais is a mono-topic membrane protein (Figure 4K, L). Given the observed mild antiphage activity of this system, it is possible that the antiphage activity is an artefact of this protein complex, perhaps causing premature lysis at a stage when phage particles are not fully matured (due to incomplete assembly or DNA packaging). However, a lipoprotein-containing antiphage system (PD-Lambda-6) has been reported previously (21).

The N-terminus of Rugutis contains a SEC-C motif based on HHpred analysis (PDB ID 2I9W). AlphaFold2 comparison of Rugutis and SEC-C motif containing protein 2I9W reveals that approximately 20 residues at the N-terminus of Rugutis superimpose well with the C-terminus of the SEC-C motif containing protein, while the remainder of the proteins do not share any similarity. Despite the high-quality AlphaFold2 score of the Rugutis structure prediction (Figure 4M), Dali analysis did not yield significant hits. Rugutis, therefore, possesses a structure that is distinct from those present in the Protein Data Bank.

For Hesat, no domain was predicted by HHpred and InterPro. Dali analysis reports significant hits with the colicin-D toxic domain (PDB ID 1V74; Figure 4N, O), which specifically cleaves the anticodon loop of all four tRNA(Arg) iso-acceptors, thereby resulting in cell death (68). Therefore, it is proposed that Hesat upon phage infection is activated to block translation, which is in accordance with its abortive infection phenotype.

Domain and structure prediction of previously described systems

InterPro predictions confirmed that the lactococcal PARIS system consists of an ATPase Associated with a variety of cellular Activities (AAA_21) and a DUF4435 domain, in accordance with the previously described PARIS system (20). Here, based on Alphafold2 prediction we provide the relevant structures (Supplementary Figure S1) and we report in more detail that the first component of the PARIS system (PARIS-i) resembles a DNA double-strand break repair Rad50 ATPase (PDB ID 3QKU: Supplementary Figure S1A, B). This supports the notion that this protein is responsible for sensing invading phage and then activating an associated nuclease by its ATPase activity via a mechanism similar to the Mre11-Rad50 homolog SbcCD (69), as has previously been suggested for the Lamassu system (19). Based on HHpred, Rousset et al., identified a weak but statistically significant match of part of the second component of PARIS (PARIS-ii) with a Topoisomerase-primase (TOPRIM) domain of OLD family nucleases (20), consistent with our Alphafold2 prediction (PDB ID 6P74; Supplementary Figure S1B, C). The lactococcal PARIS system was found to exhibit low amino acid similarity with the two distinct E. coli PARIS-1 and -2 systems (≤32%) (20). Nevertheless, AlphaFold2 predictions showed that the two systems superimpose well (Supplementary Figure S1C, F).

HHpred, InterPro and AlphaFold2/Dali revealed the presence of CBASS-related domains (70–72) in type I and II CBASS (Table 1, Supplementary Figure S2, (19)). A lactococcal type II CBASS system has previously been described, although its antiphage activity was not explored (71). No lactococcal equivalent of type I CBASS has been reported in literature to our knowledge.

Based on InterPro the lactococcal Lamassu was found to adhere to the described type II Lamassu of the recently described Lamassu family consisting of a Cap4 endonuclease, an unknown protein and a structural maintenance of the chromosomes (SMC) protein (19,39,63). Here, AlphaFold2 was able to predict the structures of Lamassu-i, -ii and -iii (Supplementary Figure S3A, C, E) as well as a hexameric complex by a dimer of the Lamassu-i, -ii, -iii trimer (Supplementary Figure S3G, H). The second component of Lamassu (Lamassu-ii or LmuC) is believed to be necessary for defensive activity, though its function was not defined (19,39). AlphaFold2/Dali predict that the D1 domain of this protein resembles a contact-dependent growth inhibition (CDI) DNAse (PDB ID 4G6U; Supplementary Figure S3D). Bacterial CDI proteins are known to act as toxins or immunity systems (73), suggesting that this is (part of) the effector of the system. Additionally, the predicted Lamassu-iii (LmuB) globular domain (D1, Supplementary Figure S3E) is similar to a dsDNA break repair Rad50 ATPase (PDB 7YZO; Supplementary Figure S3F), supporting previous reports indicating that this protein is responsible for sensing invading phage DNA upon which it activates an associated nuclease (LmuA) by its ATPase activity (19,63).

InterPro yielded hits with previously reported Septu-related proteins (ATPase and HNH endonuclease (17) for the lactococcal Septu antiphage system. AlphaFold2 was able to predict the structures of the two Septu components (Supplementary Figure S4A, B), providing novel domain information. Septu-i and -ii contain three domains and AlphaFold2 suggests that domain D3 from Septu-i binds D2 from Septu-ii (Supplementary Figure S4C, D). Dali analysis reported hits between Septu-i second domain (D2) and an excinuclease ABC (PDB ID 2VF7; Supplementary Figure S4E). The first Nt domain (D1) of Septu-ii is similar to an HNH endonuclease (PDB ID 5H0O) according to Alphafold2 and Dali, while its third domain (D3) C-terminal domain resembles a GAF domain (PDB ID 8DGD; Supplementary Figure S4F).

AbiD, AbiD1 and AbiF share modest amino acid similarity (51–53). Here, we predicted the structures of AbiD, AbiD-like, AbiD1 and AbiF (Figure 5A–D). The core of the four proteins superimpose well (Figure 5E). Furthermore, AbiD superimposes well with a Cas13d RNA-guided ribonuclease (PDB ID 6IV8; Figure 5F), including its catalytic site (residue His 293 with AbiD His520) previously associated with abortive infection (74). These results allow amalgamation of these previously distinct systems into a single Abi group and suggest a similar mode of antiphage action. We propose this antiphage Abi group henceforth to be named AbiD/F. In addition, AbiD-like will from hereon be referred to as AbiD/F.

Figure 5. Predicted structures of AbiD-like and related Abi systems. (A–D) Ribbon structure of AbiD (A), AbiD-like (B), AbiD1 (C) and AbiF (D) (rainbow coloured). (E) Superimposition of AbiD (blue), AbiD-like (green), AbiD1 (orange) and AbiF (pink). (F) Superimposition of AbiD (blue) and PDB 6IV8 (grey), a Cas13d RNA-guided ribonuclease (Z = 8.0; rmsd = 4.3 Å). (G) Molecular surface of AbiD in contact with that of the RNA from the Cas13d complex. Note the complementarity of the surfaces indicating conservation of the catalytic site. Cas13d catalytic residue His 293 superimposes with AbiD His 520. pLDDT values and predicted aligned errors (PAE) explanation present in Supplementary Figure S5.

Distribution and conjugation potential of the identified systems

Interestingly, the majority of the confirmed antiphage systems (15 out of 20) are rather rare among the gene families within our collection with only a few homologues present (≤3 gene family members/homologues, Supplementary Table S2). In contrast, Rugutis (n = 4), Hesat (n = 7), AbiF (n = 5), AbiD/F (AbiD-like, n = 9) and AbiB-like (n = 31) are more abundant gene families. It is worth noting that despite the high occurrence of AbiB homologues, only one out of the 11 (randomly picked from the 31) tested candidates (94% sequence identity to AbiB) was shown to possess antiviral activity in this study. The remaining homologues share a sequence identity level ranging from 90% to 100% compared to AbiB, with nearly half of them being full-length while the remainder apparently representing truncated versions (with coverage ranging from 16% to 88%). To determine the prevalence of the identified antiphage systems a BLASTP and HMMER search was conducted; obtained hits are listed in Table 2. BLASTP primarily reveals the presence of highly homologous proteins in other lactic acid bacteria (LAB). However, by using a more sensitive search (HMMER), homologues of these systems can be found in many different microorganisms, although their association with antiphage activity still requires verification.

Table 2. Prevalence of the discovered antiphage systems. The BLASTP hits represent the total of distinct hits with a cut-off value of > 70% amino acid identity with an associated E-value of < 0.001. The HMMER organisms represent the top two obtained HMMER hits as an example

	System name	BLASTP hits	BLASTP organisms	HMMER hits	HMMER organisms	
New antiphage systems	Rhea	8	Lactobacillales	611	Kosmotoga sp., Trichocoleus sp.	
	Aristaios	4	L. lactis, L. cremoris	≥10000	Burkholderia cepacia, Geobacillus sp.	
	Kamadhenu	8	L. lactis, L. cremoris	914	Klebsiella pneumoniae, Undibacterium rivi	
	Fliodhais-i	5	L. lactis, L. cremoris, Lactococcus sp.	5	L. cremoris, L. petauri	
	Fliodhais-ii	47	L. lactis, L. cremoris, L. garvieae, L. petauri	415	Lactobacillus sp., Lactiplantibacillus fabifermentans	
	Audmula	2	L. lactis, Enterococcus sp.	91	Enterococcus sp., Enterococcus lemanii	
	Rugutis	3	L. lactis, Lactococcus sp.	6156	Clostridiales bacterium, Pontibacillus litoralis	
	Hesat	18	Lactococcus spp., Streptococcus thermophilus, Lysinibacillus sp.	111	Streptococcus varani, L. lactis	
Systems new in Lactococcus/ -like systems	PARIS-i	4	L. piscium, L. lactis	9988	Pseudomonas qingdaonensis, Endozoicomonas acroporae	
	PARIS-ii	2	L. piscium, L. lactis	955	Morganella morganii, Reichenbachiella agariperforans	
	Type I CBASS-i	2	L. petauri, Lactococcus sp.	985	Bacteroidales, Lewinella sp.	
	Type I CBASS-ii	5	L. lactis, L. petauri, Streptococcus infantarius, Culicoidibacter larvae	6684	Campylobacter concisus, Endozoicomonas gorgoniicola	
	Type II CBASS-i	2	L. lactis	115	Clostridium liquoris, Lachnospira eligens	
	Type II CBASS-ii	2	L. lactis	4300	Saprospirales, Deltaproteobacteria	
	Type II CBASS-iii	7	Streptococcaceae	916	Bacillus sp., Desulfosporosinus metallidurans	
	Lamassu-i	1	L. lactis	540	Streptococcus sp., Pseudomonas spp.	
	Lamassu-ii	2	L. lactis	461	Staphylococcus schleiferi, Paenibacillus sp.	
	Lamassu-iii	4	L. lactis, Streptococcus salivarius, Streptococcus sanguinis	9997	Bacillus sp., Terribacillus saccharophilus	
	Septu-i	1	Lactococcus sp.	9990	Candidatus Brocadiales, Marinilabiliaceae	
	Septu-ii	1	Lactococcus sp.	5797	Streptococcus suis, Lactococcus spp.	
	AbiD/F (AbiD-like)	7	L. cremoris, Lactococcus sp.	8585	Caloramator proteoclasticus, Paenibacillus thermoaerophillus	

Finally, most of the antiphage systems (13 out of 20) described in this work are encoded either on a pMRC01- or pNP40-like conjugative plasmid (Supplementary Table S3, Supplementary Figure S6) or they are carried by a strain possessing either of these plasmid-encoded conjugation mechanisms, implying that they could either be conjugated or co-mobilized to other strains (75,76) (Supplementary Table S3). With the exception of Kamadhenu, PARIS, type II CBASS, AbiA, AbiD/F and AbiJ, the identified systems do not appear to be co-localized with other known or herein described systems (when a window of five genes upstream/downstream was employed).

Discussion

The current study constitutes a considerable contribution to knowledge of the bacterial immunity against phages by identifying seven novel antiphage systems and five known systems whose functionality had not previously been demonstrated in Lactococcus. Although recent studies have focused on genomic co-localization with known systems for the discovery of new antiphage systems (18–20), it now appears evident that many antiphage systems remain undetected as they are not necessarily located close to other such systems (Supplementary Table S3). This is also supported by a recent study (21), where the authors reported that only three out of the 21 discovered systems are situated within a defence island. Additionally, previous studies have not systematically and deliberately searched for plasmid-encoded antiphage systems. Lactococcal plasmid-associated genes are mostly of unknown function and, as phages represent the main bacterial enemy, it was hypothesized that some of these genes of unknown function are associated with antiphage activity. Indeed, one in ten of the candidate systems in this study was ultimately confirmed as an antiphage system, while this may be an underestimation of the actual number of antiphage systems present in this lactococcal strain collection as outlined below.

DefenseFinder and PADLOC were used for the detection of antiphage systems that had previously been found in bacterial species beyond L. lactis/cremoris. Even though five systems detected by these two bioinformatic tools were validated, four others did not show any antiphage effect. This lack of activity may be explained by the employment of an insufficiently diverse panel of phages, because the current phages have evolved to overcome these systems or due to incorrect testing conditions or by the possibility that these systems have been inactivated in Lactococcus over time. This could be explained by the elimination of the relevant phage(s) that these systems target or due to the cell fitness cost of maintaining an antiphage system (60,61). Furthermore, members of the AbiD/F family were shown to require an upstream sequence in addition to the genes for their antiphage activity. Therefore, candidates that did not display antiphage activity could have been false negatives since cloning with native regulatory factors was required or the system is composed of multiple genes (the current study was primarily focused on single or two component candidate antiphage systems). In addition, the many apparently inactive AbiB-like candidates demonstrate that not all members of the same family display the same activity. Therefore, given that a single representative gene was assessed and potentially selected as candidate for most of the gene families, antiphage systems may have been overlooked if an inactive or inappropriate (in terms of activity against the tested phage) member had been selected.

Interestingly, the majority of the systems tested in this study are highly effective against phages of the genus Skunavirus, which are the most commonly isolated and problematic phages in the dairy environment, suggesting that the lactococcal phage resistance have expectedly evolved to target the most prevalent and abundant lactococcal phages. The resistance phenotype was not only observed on solid, but also in liquid medium, underlining the biotechnological relevance as Lactococcus spp. are applied in the fermentation of milk. All studied systems were found to be active post-phage DNA injection and with the exception of the newly identified Audmula all tested systems appear to be consistent with an Abi phenotype. CBASS (72), Lamassu (63) and PARIS systems (20) are known to present Abi phenotypes; however, to our knowledge, this has not previously been determined for Septu. Although lysis-in-broth is still widely used to assign systems as Abi (19,21), it has been reported that the mechanistic characterization of a system as Abi-should not be deduced by the associated (phenotypic) lysis profile (59). Therefore, additional research is required to unravel the mode of action of the systems and thus assignment of the system as a validated Abi.

The newly discovered antiphage systems in this study were shown to be rare among our lactococcal plasmid collection, yet (homologues) seem to be widespread among different bacterial phyla. In addition, almost half of the 23 known lactococcal Abi systems were not found within the 1020 analysed gene families derived from 53 different strains. This indicates that either some systems are less prevalent than others and/or denoting that our strain collection and derived plasmid library do not represent the full diversity of lactococcal strains that exists. Indeed, it has been reported that most of the known antiphage systems (>50%) are found in a small proportion of bacterial genomes (<3%) with rare systems being the main diversity contributors in antiviral arsenals (22). Additionally, there are possible applications for the discovered systems regardless of their distribution considering that bacterial antiphage systems are now used as biotechnological/biomedical tools (77,78).

Despite the identification of several and diverse domains by HHpred and InterPro for most of the new systems, none of the hits were with proteins known to be involved in phage resistance. AlphaFold2 (AF2) has revolutionized structural biology by allowing accurate protein structure and complexation prediction (79,80). Indeed, further functional and structural analysis of the antiphage systems using AF2 and the fold comparator Dali revealed possible modes of action for several of the newly identified systems (46), linking Aristaios, Kamadhenu and Hesat to proteins related to programmed cell death, which is consistent with an abortive infection mode of action. Therefore, AF2 along with Dali could be applied to elucidate the possible functions, structures and complexes of emerging antiphage systems, as the mode of action of more than half of the reported systems (58%) remains unknown (37). However, these hypothetical modes of action require experimental verification to classify the systems based on their molecular mechanism (22).

The findings of this study led to the grouping of the previously distinct AbiD, AbiD1, AbiF (and AbiD/F), which share sequence similarity and superimpose structurally into a single AbiD/F group of antiphage systems. Members of this group structurally resemble the endonuclease domain of the RNA-guided RNase Cas13, which has previously been associated with abortive infection (72). Therefore, we foresee that structural analysis followed by structural alignments of the existing bacterial antiphage systems may assist in their function- and similarity-based classification. Meanwhile, structural searches of proteins of unknown function against an antiphage system database may indeed facilitate discovery of previously unknown antiphage systems.

The discovery of plasmid-encoded antiphage systems has clear implications for the dairy industry. The constant increase of the global dairy (https://www.statista.com/statistics/502280/global-dairy-market-value/) and fermented (https://www.statista.com/statistics/1033912/market-value-of-fermented-food-ingredients/) food products market and the persistent threat of phages in the production of fermented foods (81), necessitates the direct selection of strains encoding a range of antiphage systems or the improvement of phage robustness of existing strains through conjugation or other natural DNA transfer approaches.

The recent surge in discovery of previously unknown antiphage systems and their varying modes of action has greatly improved our understanding of phage-host interactions and phage resistance. Our study provides further evidence on the diversity of the antiphage systems and the significance of plasmids in acquisition of bacterial immunity against phage attack. We expect that future studies will use plasmids as a rich resource for the discovery of unknown antiphage systems in other species and will further confirm the significance of plasmids on the immune system of bacteria. While there is still much to be learnt and understood, we envision continued discovery of the complex bacterial immunity landscape, with the generated knowledge being applied in the food, biotechnology, biomedical industry and beyond.

Supplementary Material

gkae671_Supplemental_Files

Acknowledgements

We thank the D.v.S. and J.M. laboratory members for technical assistance during the design of the experiments and discussion of results. We thank Gavin Dillon and Alison Kearns for assistance during molecular cloning. The graphical abstract was created with BioRender.com.

Author contributions: A.G., C.M., J.M. and D.v.S. led the study. A.G. and C.M. performed the experiments and the analyses unless otherwise indicated. C.C. performed the AlphaFold2 predictions and analysis. M.B. contributed to the discovery and the preliminary analysis of some antiphage systems. P.K. was responsible for the genomic assembly and annotation of the lactococcal plasmid sequences and the grouping of the plasmid genes into families. B.M. contributed to the generation of some constructs and to the initial project administration. P.d.W., I.v.R. and N.v.P. contributed to meaningful discussion and interpretation of the results. J.M. and D.v.S. supervised and examined the results of the experiments. J.M. and D.v.S. conceived the project and contributed to the selection of candidate systems. The initial concept manuscript was written by A.G., C.M. and C.C., and all authors contributed to the final version of the manuscript.

Data availability

Sequences were deposited in GenBank under the accession number PRJNA1086404. Coordinates of predicted structures are accessible on Zenodo (https://doi.org/10.5281/zenodo.11221414). The structures are also presented in the main and supplementary figures. Any additional information required to reanalyse the data reported in this study is available from the corresponding author upon request.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

Science Foundation Ireland co-funded by dsm-firmenich [12/RC/2273‐P1, 12/RC/2273‐P2, 17/SP/4678]; HPC resources from GENCI-IDRIS [2023-AD010714075, in part]; for the molecular graphics we acknowledge UCSF ChimeraX which is developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from National Institutes of Health [R01-GM129325]; Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Funding for open access charge: Science Foundation Ireland [12/RC/2273‐P1, 12/RC/2273‐P2, 17/SP/4678].

Conflict of interest statement. P.d.W., I.v.R. and N.v.P. are employees of dsm-firmenich. Patent Applications (EP23166918 and EP2024/058584) have been filed in the context of this work.
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