Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in Streptococcus thermophilus (Sth) phages, a more comprehensive understanding of Acr diversity in Sth phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A Sth CRISPR-Cas systems. These acr genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage–host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.
Get full access to this article
View all access options for this article.
References
1.
MarraffiniLA. CRISPR-Cas immunity in prokaryotes. Nature, 2015; 526(7571):55–61.
2.
MakarovaKS, WolfYI, AlkhnbashiOS, et al.An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol, 2015; 13(11):722–736.
3.
HilleF, RichterH, WongSP, et al.The biology of CRISPR-Cas: Backward and forward. Cell, 2018; 172(6):1239–1259.
4.
CharpentierE, RichterH, Van Der OostJ, et al.Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiol Rev, 2015; 39(3):428–441.
5.
BrounsSJJ, JoreMM, LundgrenM, et al.Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 2008; 321(5891):960–964.
6.
WatsonBNJ, VercoeRB, SalmondGPC, et al.Type I-F CRISPR-Cas resistance against virulent phages results in abortive infection and provides population-level immunity. Nat Commun, 2019; 10(1):5526.
7.
JohnsonKA, GarrettSC, Noble-MolnarC, et al.Selective degradation of phage RNAs by the Csm6 ribonuclease provides robust type III CRISPR immunity in Streptococcus thermophilus. Nucleic Acids Res, 2024; 52(20):12549–12564.
8.
BraddeS, NourmohammadA, GoyalS, et al.The size of the immune repertoire of bacteria. Proc Natl Acad Sci U S A, 2020; 117(10):5144–5151.
9.
LevinBR, MoineauS, BushmanM, et al.The population and evolutionary dynamics of phage and bacteria with CRISPR–Mediated immunity. PLoS Genet, 2013; 9(3):e1003312.
10.
MurtazalievaK, MuA, PetrovskayaA, et al.The growing repertoire of phage anti-defence systems. Trends Microbiol, 2024; 32(12):1212–1228.
11.
StanleySY, MaxwellKL. Phage-Encoded Anti-CRISPR defenses. Annu Rev Genet, 2018; 52(1):445–464.
12.
LiY, Bondy-DenomyJ. Anti-CRISPRs go viral: The infection biology of CRISPR-Cas inhibitors. Cell Host Microbe, 2021; 29(5):704–714.
13.
Bondy-DenomyJ, PawlukA, MaxwellKL, et al.Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature, 2013; 493(7432):429–432.
14.
DavidsonAR, LuW-T, StanleySY, et al.Anti-CRISPRs: Protein inhibitors of CRISPR-Cas systems. Annu Rev Biochem, 2020; 89(1):309–332.
15.
Bondy-DenomyJ, DavidsonAR, DoudnaJA, et al.A unified resource for tracking Anti-CRISPR names. Crispr J, 2018; 1(5):304–305.
16.
YangL, ZhangY, YinP, et al.Structural insights into the inactivation of the type I-F CRISPR-Cas system by anti-CRISPR proteins. RNA Biol, 2021; 18(sup2):562–573.
17.
AllemailemKS, AlmatroudiA, AlrumaihiF, et al.current updates of CRISPR/Cas system and Anti-CRISPR proteins: Innovative applications to improve the genome editing strategies. Int J Nanomedicine, 2024; 19:10185–10212.
18.
ThavalingamA, ChengZ, GarciaB, et al.Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2. Nat Commun, 2019; 10(1):2806.
19.
TrostCN, YangJ, GarciaB, et al.An anti-CRISPR that pulls apart a CRISPR–Cas complex. Nature, 2024; 632(8024):375–382.
20.
JiangF, LiuJ-J, OsunaBA, et al.Temperature-Responsive competitive inhibition of CRISPR-Cas9. Mol Cell, 2019; 73(3):601–610.
21.
PengR, XuY, ZhuT, et al.Alternate binding modes of anti-CRISPR viral suppressors AcrF1/2 to Csy surveillance complex revealed by cryo-EM structures. Cell Res, 2017; 27(7):853–864.
22.
PonsBJ, van HouteS, WestraER, et al.Ecology and evolution of phages encoding anti-CRISPR proteins. J Mol Biol, 2023; 435(7):167974.
23.
StanleySY, BorgesAL, ChenK-H, et al.Anti-CRISPR-Associated proteins are crucial repressors of Anti-CRISPR transcription. Cell, 2019; 178(6):1452–1464.
24.
YinY, YangB, EntwistleS. Bioinformatics identification of Anti-CRISPR loci by using homology, guilt-by-association, and CRISPR self-targeting spacer approaches. mSystems, 2019; 4(5):e00455–e00419.
25.
HaoM, CuiY, QuX. Analysis of CRISPR-Cas system in Streptococcus thermophilus and its application. Front Microbiol, 2018; 9:257.
26.
PhilippeC, MoineauS. The endless battle between phages and CRISPR-Cas systems in Streptococcus thermophilus. Biochem Cell Biol, 2021; 99(4):397–402.
27.
CarteJ, ChristopherRT, SmithJT, et al.The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis Streptococcus thermophilus. Mol Microbiol, 2014; 93(1):98–112.
28.
LeprinceA, LefrançoisJ, MagillD, et al.Strengthening Phage Resistance of Streptococcus thermophilus by Leveraging Complementary Defense Systems. 2024.
29.
HynesAP, RousseauGM, AgudeloD, et al.Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nat Commun, 2018; 9(1):2919.
30.
HynesAP, RousseauGM, LemayM-L, et al.An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9. Nat Microbiol, 2017; 2(10):1374–1380.
31.
SongG, ZhangF, TianC, et al.Discovery of potent and versatile CRISPR–Cas9 inhibitors engineered for chemically controllable genome editing. Nucleic Acids Res, 2022; 50(5):2836–2853.
32.
PastuszkaA, RousseauGM, SomervilleV, et al.Dairy phages escape CRISPR defence of streptococcus thermophilus via the anti-CRISPR AcrIIA3. Int J Food Microbiol, 2023; 407:110414.
33.
EgorovAA, HauryliukV, AtkinsonGC. Systematic annotation of hyper-variability hotspots in phage genomes and plasmids. 2024.
34.
JohnsonKA, GoswamiH, CatchpoleRJ, et al.A phage-encoded anti-CRISPR protein cooperates with host enolase to prevent type III CRISPR immunity. 2025.
35.
Pinilla-RedondoR, ShehreenS, MarinoND, et al.Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements. Nat Commun, 2020; 11(1):5652.
36.
GeorjonH, BernheimA. The highly diverse antiphage defence systems of bacteria. Nat Rev Microbiol, 2023; 21(10):686–700.
37.
SilasS, CarionH, MakarovaKS, et al.Activation of bacterial programmed cell death by phage inhibitors of host immunity. Mol Cell, 2025; 85(9):1838–1851.
38.
BatemanA, MartinM-J, OrchardS, et al.UniProt: The universal protein knowledgebase in 2025. Nucleic Acids Research, 2025; 53(D1):D609–D17.
39.
ObergN, ZallotR, GerltJA. EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) web resource for genomic enzymology tools. J Mol Biol, 2023; 435(14):168018.
40.
ZallotR, ObergN, GerltJA. The EFI web resource for genomic enzymology tools: Leveraging protein, genome, and metagenome databases to discover novel enzymes and metabolic pathways. Biochemistry, 2019; 58(41):4169–4182.
41.
ShannonP, MarkielA, OzierO, et al.Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res, 2003; 13(11):2498–2504.
42.
AbramsonJ, AdlerJ, DungerJ, et al.Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024; 630(8016):493–500.
43.
HolmL, LaihoA, TörönenP, et al.DALI shines a light on remote homologs: One hundred discoveries. Protein Sci, 2023; 32(1):e4519.
44.
NishimuraY, YoshidaT, KuronishiM, et al.ViPTree: The viral proteomic tree server. Bioinformatics, 2017; 33(15):2379–2380.
45.
LetunicI, BorkP. Interactive Tree Of Life (iTOL): An online tool for phylogenetic tree display and annotation. Bioinformatics, 2007; 23(1):127–128.
46.
ZimmermannL, StephensA, NamS-Z, et al.A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J Mol Biol, 2018; 430(15):2237–2243.
47.
BlumM, AndreevaA, FlorentinoLC, et al.InterPro: The protein sequence classification resource in 2025. Nucleic Acids Res, 2025; 53(D1):D444–D456.
48.
TerzianP, Olo NdelaE, GaliezC, et al.PHROG: Families of prokaryotic virus proteins clustered using remote homology. NAR Genom Bioinform, 2021; 3(3):lqab067.
49.
van KempenM, KimSS, TumescheitC, et al.Fast and accurate protein structure search with Foldseek. Nat Biotechnol, 2024; 42(2):243–246.
50.
LévesqueC, DuplessisM, LabontéJ, et al.Genomic organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing streptococcus thermophilus strain. Appl Environ Microbiol, 2005; 71(7):4057–4068.
51.
HanemaaijerL, KelleherP, NeveH, et al.Biodiversity of phages infecting the dairy bacterium streptococcus thermophilus. Microorganisms, 2021; 9(9):1822.
52.
DuplessisM, RussellWM, RomeroDA, et al.Global gene expression analysis of two streptococcus thermophilus bacteriophages using DNA microarray. Virology, 2005; 340(2):192–208.
53.
ZhangX, GarrettS, GraveleyBR, et al.Unique properties of spacer acquisition by the type III-A CRISPR-Cas system. Nucleic Acids Res, 2022; 50(3):1562–1582.
54.
GarciaB, LeeJ, EdrakiA, et al.Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used for genome editing. Cell Rep, 2019; 29(7):1739–1746.
55.
GussowAB, ParkAE, BorgesAL, et al.Machine-learning approach expands the repertoire of anti-CRISPR protein families. Nat Commun, 2020; 11(1):3784.
56.
YiH, HuangL, YangB, et al.AcrFinder: Genome mining anti-CRISPR operons in prokaryotes and their viruses. Nucleic Acids Res, 2020; 48(W1):W358–W365.
57.
WiegandT, KarambelkarS, Bondy-DenomyJ, et al.Structures and strategies of Anti-CRISPR-Mediated immune suppression. Annu Rev Microbiol, 2020; 74(1):21–37.
58.
ZhangK, WangS, LiS, et al.Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM. Proc Natl Acad Sci USA, 2020; 117(13):7176–7182.
59.
GabelC, LiZ, ZhangH, et al.Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14. Nucleic Acids Res, 2021; 49(1):584–594.
60.
StanleyE, FitzgeraldGF, MarrecCL, et al.Sequence analysis and characterization of φO1205, a temperate bacteriophage infecting Streptococcus thermophilus CNRZ1205. Microbiology (Reading), 1997; 143(11):3417–3429.
61.
BruttinA, FoleyS, BrüssowH. The site-specific integration system of the temperate streptococcus thermophilus bacteriophage φSfi21. Virology, 1997; 237(1):148–158.
62.
ChowdhuryS, CarterJ, RollinsMF, et al.Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-Guided surveillance Complex. Cell, 2017; 169(1):47–57.
63.
PhilippeC, MorencyC, PlanteP-L, et al.A truncated anti-CRISPR protein prevents spacer acquisition but not interference. Nat Commun, 2022; 13(1):2802.
64.
WattersKE, ShivramH, FellmannC, et al.Potent CRISPR-Cas9 inhibitors from staphylococcus genomes. Proc Natl Acad Sci USA, 2020; 117(12):6531–6539.
65.
LiuL, YinM, WangM, et al.Phage AcrIIA2 DNA mimicry: Structural basis of the CRISPR and Anti-CRISPR arms race. Mol Cell, 2019; 73(3):611–620.
66.
YangH, PatelDJ. Inhibition mechanism of an Anti-CRISPR suppressor AcrIIA4 targeting SpyCas9. Mol Cell, 2017; 67(1):117–127.
67.
Moura de SousaJA, PfeiferE, TouchonM, et al.Causes and consequences of bacteriophage diversification via genetic exchanges across lifestyles and bacterial Taxa. Mol Biol Evol, 2021; 38(6):2497–2512.
68.
YangH, MaY, WangY, et al.Transcription regulation mechanisms of bacteriophages. Bioengineered, 2014; 5(5):300–304.
69.
MaraP, VikD, PachiadakiMG, et al.Viral elements and their potential influence on microbial processes along the permanently stratified Cariaco Basin redoxcline. Isme J, 2020; 14(12):3079–3092.
70.
Van Den BergB, SilaleA, BasléA, et al.Structural basis for host recognition and superinfection exclusion by bacteriophage T5. Proc Natl Acad Sci USA, 2022; 119(42).
71.
CumbyN, DavidsonAR, MaxwellKL. The moron comes of age. Bacteriophage, 2012; 2(4):e23146.
72.
AliY, KobergS, HeßnerS, et al.Temperate Streptococcus thermophilus phages expressing superinfection exclusion proteins of the Ltp type. Front Microbiol, 2014; 5:98.
73.
AdamsDW, WuLJ, ErringtonJ. Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane. Embo J, 2015; 34(4):491–501.
74.
MohantyBK, KushnerSR. Ribonuclease P processes polycistronic tRNA transcripts in Escherichia coli independent of ribonuclease E. Nucleic Acids Res, 2007; 35(22):7614–7625.
75.
LiuY, ZhangL, GuoM, et al.Structural basis for anti-CRISPR repression mediated by bacterial operon proteins Aca1 and Aca2. J Biol Chem, 2021; 297(6):101357.
76.
BirkholzN, KamataK, FeussnerM, et al.Phage anti-CRISPR control by an RNA- and DNA-binding helix–turn–helix protein. Nature, 2024; 631(8021):670–677.
77.
GongC, ChakrabortyD, KoudelkaGB. A prophage encoded ribosomal RNA methyltransferase regulates the virulence of Shiga-Toxin-Producing Escherichia Coli (STEC). Nucleic Acids Res, 2024; 52(2):856–871.
78.
ThompsonLR, ZengQ, KellyL, et al.Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism. Proc Natl Acad Sci USA, 2011; 108(39):E757–E764.
79.
HederstedtL. Diversity of cytochrome c oxidase assembly proteins in bacteria. Microorganisms, 2022; 10(5):926.
80.
CheungVWN, XueB, Hernandez-ValladaresM, et al.Identification of polyketide inhibitors targeting 3-Dehydroquinate dehydratase in the shikimate pathway of enterococcus faecalis. PLoS One, 2014; 9(7):e103598.
81.
RichterSG, ElliD, KimHK, et al.Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria. Proc Natl Acad Sci USA, 2013; 110(9):3531–3536.
82.
ZhangK, LiS, WangY, et al.Bacteriophage protein PEIP is a potent Bacillus subtilis enolase inhibitor. Cell Rep, 2022; 40(1):111026.
83.
WangX, LiX, MaY, et al.Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18. Nucleic Acids Res, 2022; 50(1):512–521.
84.
AchigarR, MagadánAH, TremblayDM, et al.Phage-host interactions in Streptococcus thermophilus: Genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array. Sci Rep, 2017; 7(1):43438.
85.
Le MarrecC, van SinderenD, WalshL, et al.Two groups of bacteriophages infecting streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins. Appl Environ Microbiol, 1997; 63(8):3246–3253.
SamuelB, MittelmanK, CroitoruSY, et al.Diverse anti-defence systems are encoded in the leading region of plasmids. Nature, 2024; 635(8037):186–192.
88.
RoucourtB, LavigneR. The role of interactions between phage and bacterial proteins within the infected cell: A diverse and puzzling interactome. Environ Microbiol, 2009; 11(11):2789–2805.
89.
BorgesAL, DavidsonAR, Bondy-DenomyJ. The discovery, mechanisms, and evolutionary impact of Anti-CRISPRs. Annu Rev Virol, 2017; 4(1):37–59.
90.
KelleherP, Ortiz CharnecoG, KampffZ, et al.Phage defence loci of streptococcus thermophilus tip of the anti-phage iceberg? Nucleic Acids Res, 2024; 52(19):11853–11869.
91.
KooninEV, MakarovaKS, WolfYI. Evolutionary genomics of defense systems in archaea and bacteria. Annu Rev Microbiol, 2017; 71(1):233–261.
92.
Bhoobalan-ChittyY, XuS, Martinez-AlvarezL, et al.Regulatory sequence-based discovery of anti-defense genes in archaeal viruses. Nat Commun, 2024; 15(1):3699.
93.
HuT, CuiY, QuX. Characterization and comparison of CRISPR Loci in Streptococcus thermophilus. Arch Microbiol, 2020; 202(4):695–710.
94.
KirchbergerPC, MartinezZA, LukerLJ, et al.Defensive hypervariable regions confer superinfection exclusion in microviruses. Proc Natl Acad Sci U S A, 2021; 118(28):e2102786118.
