Bacterial Defense and Phage Counterdefense: Unraveling the Molecular Basis of Phage Resistance

Abstract

Phage therapy represents a promising strategy against multidrug-resistant bacteria, but its clinical effectiveness is increasingly compromised by the emergence of phage-resistant traits (PRTs). This resistance develops through multiple interconnected mechanisms that operate at different levels of bacterial defense. At the primary level, mutations in phage receptors, cell surface glycoconjugates (lipopolysaccharide, capsule polysaccharide, exopolysaccharide), outer membrane proteins, drug efflux pumps, and surface layer proteins provide the first line of defense by preventing phage adsorption. These receptors are highly diverse across Gram-negative bacteria, Gram-positive bacteria, and acid-fast bacteria, with PRT being particularly dominant in Gram-negative species. At the secondary level, bacteria deploy sophisticated antiphage defense (APD) systems, including CRISPR-Cas, restriction-modification systems, and abortive infection mechanisms. The recent discovery of over 150 distinct APD systems underscores the remarkable complexity of bacterial immunity. Concurrently, bacteriophages have evolved anti-antiphage defense (AAPD) systems to counter bacterial immunity, establishing a dynamic coevolutionary arms race. This continuous interplay between APD diversification and AAPD counteradaptation drives the rapid emergence of PRT, which threatens the long-term viability of phage therapy. This systematic review examines the molecular basis of phage resistance across diverse bacterial taxa, analyzes the evolutionary and coevolutionary forces shaping APD and AAPD systems, and highlights previously overlooked aspects of phage–bacteria interactions. By integrating these perspectives, we aim to inform strategies for advancing modern phage therapy and guiding future research directions.

Introduction

Bacteriophages (phages) are viruses that infect bacteria as a part of their life cycle. Lytic phages most normally lyse infected cells throughout their replication cycles. In general, the life cycles of lytic phages include phage adhesion to the bacterial cell surface, injection of the phage genome, hijacking the host cellular machinery, multiplication of phage particles, and subsequent lysis of the host bacteria to release more new phage particles. Lysogenic phages usually integrate their genetic material within the host genome. However, recent research has indicated that phages go beyond the traditional binary lytic–lysogenic lifestyle. For example, they may present with pseudolysogeny, a carrier state or a chronic infection state. Pseudolysogeny is a state in which the genomic content of the phage resides inside the host bacteria without effective integration or active replication. In carrier state mode, the phage genome is maintained within the bacterial population without genomic integration or immediate lysis. In the chronic infection state, phages continuously release new virions without destroying the host cell (Figure ).

1.

Life styles of bacteriophages. The phage genomes are represented by small dark circles, and the injected phage genome is represented by a black spring-like line. The bacterial genomes are represented by large green circles, where n is the number of cell divisions of the host bacteria, and t is the production time of new virions. In the chronic infection state, new virions are spontaneously released from the host bacteria as small carrier bubbles without destroying the host cell.

The dynamic switching between different modes of life (lytic, lysogeny, pseudolysogeny, and chronic) is one way to adapt to the dynamic environment. Temperate phages also use ecological strategies such as “piggyback-the-winner” and “piggyback-the-loser”, depending on the host population density and environmental factors. Phage adhesion to a bacterial cell surface is facilitated by highly dynamic and specific interactions between phage tail fiber proteins and bacterial cell surface architectures (phage receptors). , Lytic phages inspire phage therapy against emerging multidrug-resistant bacteria. However, the rise of PRT challenges current phage therapy. Understanding the reasons behind the emergence of PRT is one way to keep phage therapy effective. The genomic plasticity and integrity of bacteria are most often challenged by invading “nonself” genetic materials, which offer considerable evolutionary training to the bacterial community. Additionally, invading “nonself” genetic materials are the primary driving force for the emergence and diversification of APD systems, which are part of the reason for the emergence of PRT. The CRISPR-Cas, restriction modification (R–M) system, abortive infection (Abi) system, AVAST, DISARM, and CRISPR-repressed toxin–antitoxin system are among the few examples of the most common APD systems effective against diverse phages. There are more than one hundred and 50 diverse APDs known to date. However, phages simultaneously evolve to evade the APD mechanisms of bacteria via AAPD systems.

In this systematic review article, we included high-quality, original research articles on the “phage resistance” trait and evolved “antiphage” signaling. Mutations of diverse types in diverse phage receptors, including cell surface glycoconjugates, outer membrane proteins, and drug efflux pumps (EPs), lead to the emergence of PRT along with mutations in enzymes involved in amino acid metabolism, environmental adaptability, and regulatory genetic components. Phage–antibiotic synergy (PAS) has also significantly contributed to the evolution of PRT, as some phages use various EPs as their main receptors for infection. Notably, phage-driven modifications of EP or complete loss of EP expression contributed to the emergence of PRT, mostly with increased antimicrobial drug sensitivity (ADS). In addition to mutational events in the host genome, PRT has emerged as a result of several highly diverse new mechanisms, including the modulation of K⁺ pumps and the maintenance of insufficient cellular levels of Leu^TAA tRNA. Notably, PRT is dominant in Gram-negative bacteria. We believe that this review may be helpful for improving the understanding of the emergence of PRT, which may direct the advancement of phage therapy.

This review is organized to first examine resistance mechanisms at the bacterial surface (phage receptors), followed by investigations of intracellular antiphage defense systems and phage counterdefense strategies, and finally, a synthesis of additional factors contributing to phage resistance.

Methods

A literature search was performed via previous methods with the following keyword combinations: phage resistant, bacteriophage, and phage antibiotic synergy. It includes original research articles on the “phage resistance” trait and evolved “antiphage” signaling.

Defensive Strategies of Bacteria against Bacteriophages

Bacteria constantly evolve themselves and are equipped with diverse defense mechanisms to protect themselves from phage predation. Phage receptors are highly diverse cell surface architectures that participate in highly dynamic, specific interactions with bacteria. In bacteria, mutations in phage receptor genes, receptor-associated genes, and APD systems provide defense against phage infections and direct the emergence of PRT as part of genome evolution.

As part of antiphage strategies, bacterial genomic evolution and training are mediated mainly by a group of sensor and effector proteins that eliminate the entire phage-infected host cell (e.g., the Abi system) or destroy the genomic and proteineous components of the invading phage species. These sensor and effector proteins are essential components of the APD systems of bacteria, and they are extensively diverse. APD systems usually reside within the bacterial genome or may be carried within extrachromosomal genetic materials (e.g., plasmids).

In addition to classical systems such as CRISPR-Cas and restriction-modification, recent large-scale bioinformatic analyses have identified over 150 distinct antiphage defense systems, many of which are clustered in genomic “defense islands”. These newly discovered systems employ diverse mechanisms, including nucleotide depletion (e.g., DSR-2, SIR2-domain proteins), DNA/RNA cleavage (e.g., Gabija, Hachiman), and signaling-induced abortive infection (e.g., Thoeris, CBASS). The discovery of such extensive diversity underscores that bacterial immunity is far more complex than previously appreciated, with multiple systems often coexisting within a single genome to provide layered protection against diverse phages.

Attack against Bacteriophages

Mutations in Diverse Phage Receptors

Phage receptor diversity varies fundamentally across bacterial cell wall architectures. In this section, we examine receptor types organized by their molecular class, glycoconjugates, proteinaceous structures, and surface appendages while highlighting distinctions between Gram-negative bacteria (GNB), Gram-positive bacteria (GPB), and acid-fast bacteria (AFB) throughout.

Phage receptors vary among Gram-negative bacteria (GNB), Gram-positive bacteria (GPB), and acid-fast bacteria (AFB) on the basis of their cell wall or membrane composition. In GNB, the most common phage receptors are lipopolysaccharide (LPS), capsule polysaccharide (CPS), and exopolysaccharide (EPS). Conversely, in the GPB, wall teichoic acid (WTA) decorations and in the AFB, glycopeptidolipid (GPL) decorations facilitate highly specific, dynamic phage–bacteria interactions. In addition, various outer membrane proteins also function as primary phage receptors. For example, the outer membrane protein A (ompA), multidrug efflux pump (mdtK, mdtN), type II, III secretion system, and type IV pilus system are used. Phage adhesion to the bacterial surface sometimes relies on the presence of additional cell surface appendices such as flagella or pila. , Mutations of diverse types, including deletions, insertions, frameshifts, missesense, nonsense, single nucleotide polymorphisms (SNPs) and point mutations in phage receptor- and receptor-associated genes, significantly contributed to the emergence of PRT.

Primary Phage Receptors: Cell Surface Glycoconjugates

Lipopolysaccharide

Lipopolysaccharide is a rigid, flexible membrane anchored to amphiphilic, glycolipid molecules. It is extremely important for the structural and functional integrity of GNB. LPS is composed of membrane-anchored phospholipids, i.e., Lipid A, and an outer membrane flanking a polysaccharide chain consisting of a core region and an O-specific chain. The core region resides adjacent to the Lipid A moiety and is chemically divided into an outer core region and an inner core region. O-specific chains or O-antigens usually have highly variable structural compositions and are linked primarily to the immune response, immune evasion or pathogenicity (Figure ).

2.

Molecular architecture of the phage receptor lipopolysaccharide. (A) Successful lytic life cycle of phage with host bacteria having wild-type lipopolysaccharide (LPS^wt), (B) impaired phage adsorption with host bacteria having mutations in galU, and (C) waa. Outer membrane (OM), production time of new virions (t).

As such, the structural complexity of LPS is highly synonymous with the participation of several enzymes for complete LPS biosynthesis. LPS also functions as a primary receptor and accessory receptor along with other membrane proteins. Mutations in the genes linked to LPS biosynthesis resulted in O-antigen depletion or truncated LPS and were associated with the emergence of PRT in several clinically important GNBs, such as Escherichia coli , Haemophilus influenzae , Klebsiella pneumoniae , Pseudomonas aeruginosa , Shigella flexneri , Salmonella enterica serovar Enteritidis and Yersinia pestis. − In addition, some agriculturally important GNBs, such as Aeromonas salmonicida , Pseudomonas syringae pv tomato DC3000, A. salmonicida , and Xanthomonas oryzae pv oryzae.

Mutation in glucose-1-phosphate uridylyltransferase (galU) is linked to phage resistance across multiple Gram-negative and Gram-positive bacteria.

In GNB, galU functions as a multipurpose gene and mainly takes part in the biosynthesis of the core polysaccharide components of LPS. The UTP-glucose-1-phosphate uridylyltransferase, i.e., the product of galU, catalyzes the formation of UDP-glucose from UTP and α-d-glucose-1-phosphate, a precursor sugar for the LPS core and O-antigen synthesis. Conversely, in GPB, e.g., Listeria monocytogenes serovar, functional galU is also essential for wall teichoic acid glycosylation, and mutations in galU are linked to the emergence of PRT. In GNB, galU resides near rfaG and rfaP due to their functional closeness with LPS biosynthesis. The O-antigen gene cluster lies between the carbohydrate metabolism genes galF and gnd. Recent studies have reported that mutations in galU (Figure ) are associated with the emergence of PRT in many species, including P. aeruginosa , E. coli , S. enterica serovar Enteritidis , S. flexneri, Aeromonas salmoicida , and K. pneumoniae. ,,, In addition to galU, the rfb/waa gene cluster also participates in the LPS biosynthesis pathway, and mutations in gene candidates of the rfb/waa gene cluster contribute to the emergence of PRT (Figure ). The waa gene cluster has been well characterized in E. coli and Salmonella , and it displays significant homology across multiple GNB species.

3.

Mutation profile of glucose-1-phosphate uridylytransferase. (A) Table presenting different mutation patterns of GalU in different E. coli strains. (B) Wild-type chain A of GalU (2E3D) is presented as a cartoon with important residues labeled.

Capsule Polysaccharides

Capsular polysaccharide is a long and high-molecular-weight carbohydrate polymer that surrounds the cell surface layer of many bacterial species. In K. pneumoniae , CPS and LPS are the major factors linked to virulence properties. On cell surfaces, CPS is present as a thick and protective layer with a greater relative abundance than that of LPS. K-antigen, the outermost most common component of the CPS (Figure ), is highly variable, and more than 140 k-serotypes are known today. In many GNB species, the CPS is linked to immune evasion or the immune response, and the CPS also acts as a primary phage receptor with high specificity. For example, phage KP32 produces two capsule depolymerases, i.e., KP32gp37 and KP32gp38, with high specificity for only the K3 and K21 serotypes of K. pneumoniae. In Klebsiella sp. CPS biosynthesis and cell surface migration depend primarily on the Wzx/Wzy-dependent pathway. In GNB, mutations in the gene candidate of the cps locus and cps regulatory elements resulted in the emergence of PRT. In Klebsiella sp. wzi, wzc are embedded within conserved regions, yet a recent report indicated that PRT arises with mutated wzi and wzc. Furthermore, WbaP and WcaJ are responsible for the biosynthesis of the highly variable middle region of the CPS, even though wbaP and wcaJ gain mutations and contribute to the emergence of PRT. In addition, mutations in the response regulator proteins dcuR, protein kinase (yeaG), and phage cox protein (soj) are associated with PRT in K. pneumoniae. In E. coli , mutations in the transcriptional regulator of the colanic acid capsular biosynthesis genes rcsA, K1, K5 and K15, which encode capsular polysaccharides with deformed LPS, also resulted in the emergence of PRT. , Nonetheless, in Campylobacter jejuni , PRT emerges due to dynamic switching (ON/OFF) of cps genes such as cj1421, cj1422 and cj1426. This dynamic “ON/OFF” process resulted in the modification of the MeOPN-GalfNAc moiety of CPS and, ultimately, CPS decoration (Figure ). In GPB species, decorative polysaccharide patterns on top of the cell wall component are necessary for structural integrity and pathogenicity and are also associated with the emergence of PRT. For example, deletion of the cell wall remodeling and autolysis gene (ΔyccB) in Lactococcus lactis and mutation of the gene responsible for polysaccharide pellicle synthesis (pbp2b) also resulted in PRT. , Moreover, the insertion sequence IS903B disrupts the capsular polysaccharide biosynthesis gene wbaZ, resulting in lytic phage resistance in ST11 K64 carbapenem-resistant K. pneumoniae.

4.

Molecular structure of phage receptor capsule polysaccharide. (A) Successful lytic life cycle of phage with host bacteria having wild-type capsule polysaccharide (CPS^wt), (B) impaired phage adsorption with host bacteria having mutant-type CPS (CPS^mt), (C) dynamic ON/OFF of the MeOPN-GalfNAc operon containing genes cj1421, cj1422 and cj1426 (D) table present gene mutations linked to the CPS decoration patterns in different bacterial species, Klebsiella pneumoniae (KP), and Lactococcus lactis (LL). Outer membrane (OM), production time of new virions (t).

Exopolysaccharides, Enterococcal Polysaccharide Antigens, and Glycopeptidolipids

Microbial biofilms are highly organized microbial communities embedded in self-produced extracellular polymeric substances. The spatial organization of microbial biofilms sometimes shields microbial cells from phage exposure, which may have a profound impact on the relative advantages or disadvantages of phage resistance across multiple GNB and GPB (Figure ).

5.

Molecular structure of phage receptor exopolysaccharides and biofilm-mediated phage-bacteria signaling. (A) Successful lytic life cycle with host bacteria having wild-type variants of EPS, LPS and CPS, (B) impaired phage adsorption with host bacteria having mutations in EPS biosynthesis- and EPS biosynthesis-related genes, adjacent table presenting mutant genes linked to EPS biosynthesis in Klebsiella pneumoniae and Pseudomonas aeruginosa (PA), Escherichia coli (EC), (C) impaired phage adsorption of the NfrB-dependent phage with host bacteria having mutation or deletion of the DgcJ gene, (D) phage resistance and sensitivity dynamics mediated by a biofilm matrix with a phage having capsular depolymerase (PBD+) and a phage without capsular depolymerase (PBD-), production time of new virions (t).

As such, recent research has indicated that the biofilm structure permits the coexistence of phage-resistant and phage-susceptible strains of E. coli. Bacterial capsular polysaccharides are broken down by phage-borne depolymerases (PBDs), which mostly reside at phage tail fibers. Active PBD ensures capsule loss across the biofilm matrix. Minute modifications of phage tail fibers and associated capsular depolymerases reflect the distribution of the surface capsule and EPS and consequently contribute to the emergence of PRT. For example, unassembled phage tail fibers released from phage-lysed bacteria are associated with PRT in K. pneumoniae. Moreover, biofilms also promote rapid growth resumption and PRT development in K. pneumoniae. In carbapenem-resistant K. pneumoniae, mutations in the EPS synthesis gene epsJ and in P. aeruginosa , mutations in EPS-associated genes (ssg, pslA) − or biofilm-related genes (fapF, crgC) associated with PRT. Mutations in EPS/biofilm-related genes reduce biofilm production with poor slimy cover. Moreover, phage-resistant P. aeruginosa is hypersensitive to colistin, suggesting the presence of a synergistic mechanism between phages and antibiotics. , PRT also developed in the E. coli biofilm community. For example, in E. coli , the diguanylate cyclase DgcJ synthesizes cyclic di-GMP from 2GTP. Biofilm formation and associated virulence properties are regulated by cyclic dimeric guanosine monophosphate (c-di-GMP) (Figure ). Disruption or deletion of dgcJ resulted in biofilm depletion and N4 phage resistance in LPS mutant E. coli. DgcJ is specifically required for N4 phage infection, as it interacts directly with the c-di-GMP-binding receptor NfrB. PRT also emerges in the Bacillus subtilis strain because the disrupted biofilm operon tapA-sipW-tasA is controlled and modulated by potassium (K⁺) ion uptake. SipW works as a bifunctional signal peptidase and regulates surface-adhered biofilm formation in association with tapA. Enterococcus faecalis is an opportunistic Gram-positive pathogen that is naturally capable of robust biofilm production. In E. faecalis , Epa gene products are linked to the biosynthesis and decoration of cell surface polysaccharides. Mutations in the EpaR/E/W and Yqw genes negatively affect EPS structure (Figure ), and precise structural modifications contribute to the emergence of PRT. In Mycobacterium smegmatis , PRT arises as a result of defects in cell wall-associated glycopeptidolipid (GPL) biosynthesis, transposon-disrupted genes (gtf2) and MbtH-like proteins (mps1, mps2).

6.

Molecular structure of the different phage receptors of Gram-positive bacteria. (A) Successful lytic life cycle of phage with host bacteria having wild-type wall teichoic acid (WTA^wt), wild-type lipoteichoic acid (LTA^wt), and wild-type enterococcal polysaccharide antigen (EPA^wt); (B) impaired phage adsorption with host bacteria having mutant type teichoic acid (WTA^mt), mutant type lipoteichoic acid (LTA^mt), and mutant type enterococcal polysaccharide antigen; (C) successful lytic life cycle of phage with host bacteria having wild-type surface layer protein (SLP^wt) connected with wild-type secondary cell wall polysaccharide (SCWP^wt); (D) impaired phage adsorption with host bacteria having mutant type surface layer protein (SLP^mt) disconnected with mutant type secondary cell wall polysaccharide (SCWP^mt). Peptidoglycan layer (PG), cell membrane (CM), and production time of new virions (t).

Wall Teichoic Acid and Lipoteichoic Acid

Teichoic acids (TAs) are the primary class of anionic cell surface glycopolymers found in GPB. There are two types of TAs: lipoteichoic acid (LTA) and wall teichoic acid (WTA). LTA and WTA differ in terms of location, structure and function. WTA usually covalently attaches to the peptidoglycan layer of the cell wall and extends through and beyond cell wall components (Figure ). LTA is anchored to the cytoplasmic membrane and extends through the peptidoglycan layer without covalent attachment. In GPB, both LTA and WTA work as phage receptors, and mutations in genes linked to TA biosynthesis result in the emergence of PRT (Figure ). For example, phage-resistant L. monocytogenes serotypes 1/2a have emerged because only WTA decorations resulting in the loss of WTA rhamnose have been altered. In L. monocytogenes serovar 4b, the removal of galactose from WTA and LTA results in the emergence of PRT. Similarly, a lack of rhamnose on WTA decorations resulted in the emergence of PRT due to nonsense mutations in multiple genes, such as LMRG_00278 (encodes the acid-resistance family protein HdeD) and LMRG_01613 (encodes the foldase PrsA precursor). PRT also emerges due to a lack of terminal N-acetylglucosamine along with rhamnose deprivation on WTA. In L. monocytogenes, Salmonella serovars with WTAs are composed of repeated ribitol-phosphate (RboP) subunits, and their hydroxyl groups, with diverse monosaccharide substitutions, define different serotypes. Among the different monosaccharide substitutions, l-rhamnose (Rha) significantly contributes to antibiotic susceptibility and virulence. A B. subtilis mutation in the gtaB gene altered WTA decoration and contributed to the emergence of PRT. UTP-glucose-1-phosphate uridylyltransferase (UGPase) product of gtaB is essential for the production of UDP-glucose. UDP-glucose is a key precursor required for glucose decoration on the WTA surface and the formation of glycolipids in the bacterial cell wall.

Flagella and Pili

Flagella are well-known long helical, filamentous structures in many bacterial species and are usually divided into three parts: a membrane complex, a flexible hook, and a flagellin filament. It is primarily responsible for the bacterial motility response activated by favorable or unfavorable environmental conditions. It is also linked to virulence properties, including biofilm formation and PRT. For example, mutations in genes linked to flagellar biosynthesis are associated with the emergence of PRT in multiple bacterial species, including Cronobacter sakazakii (flgK), E. coli (fliR), Lactobacillus plantarum (fliC, fliD), P. aeruginosa (flgK, L, pilB), and S. enterica (fliC, flgE). Notably, recent research by Wang et al., 2025 demonstrated that X1 phage DNA entry is facilitated by flagellar tubes and motors, followed by activation of the flagellum-dependent toxin-antitoxin (Flag-TA) system. Moreover, the H323A point mutation obliterates the RNAase activity of the Flag-toxin, which inactivates the Flag-TA system. Many bacterial species, including E. coli, Salmonella sp., and Serrati sp., become insensitive to X1 phages because they carry mutant filC or motA. Mutants filC or motA contribute to the complete loss of flagella or incapacitate flagellar motor movements. In addition to flagella, tiny hair-like cell–surface small protein appendages and pilos are involved in a variety of critical cellular processes, including twitching motility, surface adhesion, biofilm formation, DNA uptake, pathogenesis and the emergence of PRT. Pili are of different types, including e-pili, type-iv pili (T4P), and csu pili. Recent research has indicated that typical pili and T4P facilitate phage infections in P. aeruginosa. However, Caroff et al. 2017 reported that mutations in pilus synthesis genes reduce motility but are not the primary reasons for phage resistance. T4P is a dedicated structurally dynamic surface-exposed filamentous protein appendage. In P. aeruginosa , T4P mechanochemically regulates virulence factors. The biosynthesis of T4P in P. aeruginosa is a complex biological process that involves many genes classified as major structural pilin genes, minor accessory genes, trans-envelope assembly genes, regulatory genes, and retractor and extension motor ATPases. In P. aeruginosa , mutations in T4P biosynthetic genes such as pilA, pilB, pilD, pilQ, pilS, and pilT impair phage adhesion to the bacterial surface and contribute to the emergence of PRT. ,,,,,

Outer Membrane Proteins

Outer membrane proteins (OMPs) are integral membrane proteins that decorate the outer membrane leaflet of GNB (Figure ).

7.

Phage receptor outer membrane proteins and phage–antibiotic synergy signaling. (A) Successful lytic life cycle of phage with host bacteria having wild-type outer membrane proteins; (B) impaired phage adsorption with host bacteria having a mutant variant of OMP (OMP^mt), including structural OMPs, secretion systems, porins, TonB-dependent receptors, and efflux pumps; (C) phage–antibiotic synergy signaling, wild-type drug efflux pump (EP^wt), mutant-type drug efflux pump (EP^mt), antibacterial drug sensitivity (ABS), phage sensitivity (PS), phage resistance (PR), outer membrane (OM), and production time of new virions (t).

Structurally, OMPs adopt a β-barrel architecture, perform numerous housekeeping tasks and pathogenicity and contribute to the emergence of PRT. Many phage species utilize OMPs as their primary receptor, and mutations in OMP genes are associated with the emergence of PRT in GNB (Figure ). For example, mutations in the ferric iron transporter (fhuA) and outer membrane protein A (ompA) linked to the emergence of PRT, including ompC, ompF, ompH, ompK, vitamine-B12 transporter (BtuB), type II secretion system (T2SS), type III secretion system (T3SS), two-component system (TCS), hypothetical outer membrane protein (homP), and multidrug efflux pumps such as mdtC, MexEF-OprN, MexXY-OprM, and AcrAB-TolC, also contributed to the emergence of PRT in multiple GNBs. Salmonella anatum mutation in TonB-dependent protein (btuB) is associated with the emergence of PRT. In E. coli BL21, mutations in OMPs such as FhuA, FepA, OmpF, FadL, Tsx, and YncD contribute to the emergence of PRT. Moreover, in the Brucella abortus strain Bab8416, a mutation in ompA, along with other proteins functionally related to flagellar and efflux pumps, was detected. NmpC is an outer membrane porin gene in Salmonella sp. that contributes to antibiotic resistance and is associated with PRT. Phage–antibiotic synergy (Figure ) also contributes significantly to the emergence of PRT when drug efflux pumps are utilized as phage receptors. For example, MeXY-OprM is a known antibiotic efflux pump linked to drug resistance. The bacteriophage T7 uses MeXY-OprM as a receptor for infection. The wild-type MexXY-OprM pump provides the necessary facilities for phage infection. Deletions or mutations in MexXY-OprM contributed to the emergence of PRT. However, PRT comes at the cost of reduced antibiotic efflux, which consequently increases the antibiotic susceptibility of host bacteria (Koderi Valappil S et al., 2021).

Proteins Present in the Cell Surface Layer

Bacterial cell surface layer proteins (CSPs) are linked to the peptidoglycan layer and decorate the top of the outer membrane of most GPB, and CSPs play important roles in cell signaling, including PRT. For example, deletion of the S-layer protein in Lactobacillus helveticus and structural defects in CsaB in Bacillus anthraci contributed to the emergence of PRT. In Clostridium difficile , phage resistance is also linked with the mutant cell wall protein CwpV. The surface layer homology (SLH) domains of CSPs communicate with cell surface glycoconjugates and secondary cell wall polysaccharides (SCWPs) (Figure ). The connection between S-layer proteins and bacterial surface polysaccharides such as LPS, EPS, and CPS is often mediated by specific SLH domains. These SLH domains facilitate the binding of S-layer proteins to the carbohydrate moiety of the cell envelope, including components such as teichoic acids or SCWPs. Surface plasmon resonance studies confirm that the SLH domain is solely responsible for binding to acidic polysaccharides containing sugars, e.g., N-acetylglucosamine with pyruvate modifications, thus anchoring the S-layer to the polysaccharide matrix (Ilk et al., 2004). Structural studies have shown that SLH domains form trimeric units that recognize and bind specific carbohydrate moieties on bacterial surface polysaccharides. Thus, the S-layer can attach to the underlying cell envelope matrix (Fagan et al., 2012; Kern et al., 2018) and take part in highly specific phage–bacteria interactions.

Antiphage Defense Systems

Diversity of Antiphage Defense Systems

Bacteria are constantly evolving with an extensive and varied arsenal of defense mechanisms against phage infections. There are more than one hundred and 50 highly diverse antiphage defense systems clustered in the genomic “defense island”, underscoring high complexity and diversity beyond the classical CRISPR-Cas system available at https://defensefinder.mdmlab.fr with constant updates. Moreover, evolutionary and coevolutionary forces direct and drive bacteria and phages to diversify these systems. These processes have important implications for phage therapy and microbial ecology.

Counterattack: Anti-Antiphage Defense Signaling

The antiphage defense signals of phages are counter defense measures against high diversifications of APDs over time. The mode of phage–bacteria interactions often provides evolutionary clues toward the emergence and counterdiversification of counterdefense strategies. For example, the K. pneumoniae phage ZX1Δint infects the K. pneumoniae strain 111–2 via highly specific and dynamic interactions between CPS and the primary receptor-binding protein (RBP) ORF60 and the auxiliary RBP ORF59. Mutations in major CPS biosynthesis genes (wbaP, wbaz, and wzc) provide immunity against ZX1Δint infection. Notably, adaptive mutant phages (ZX1Δin) rapidly evolve with the Q777R mutation in the auxiliary RBP (ORF59) to regain reinfection capacity in both the wild-type and mutant type K. pneumoniae strains 111–2 (Li, Ma et al. 2025). Phage-induced antibiotic sensitivity (PIAS) often manifests in host bacteria when a multidrug-resistant efflux pump is utilized as a phage receptor. Phage-induced antibiotic-sensitive host bacteria carry a compromised multidrug efflux system (MexXY-OprM) due to phage-induced mutations in the bacterial genome (Figure ). These mutations include SNPs or large genomic deletions and direct emergence of PRT. The phage coevolutionary process also promotes mutations in phage tail fiber proteins, restoring the infection capacity of the phages.

Counterdefense Signaling against the Abortive Infection Defense System

The abortive infection (Abi) system is a unique self-destructive defense strategy of bacteria against bacteriophage infections (Figure ).

8.

Abortive infection defense and phage counterdefense signaling. Bacteria employ abortive infection (Abi) systems that trigger self-destruction upon phage infection to limit viral spread (A). Phages counteract these defenses through diverse mechanisms: DSAD1 and “nip” inhibit NADase-based defenses (B,C); TifA redirects ToxIN activity (D); AdfB abrogates DarTG toxin activity (E); GAD1/GAD2, Had1, Tad2, and Acb1/Apyc1 block DNA-cleaving (Gabija, Hachiman), signaling (Thoeris), and cyclic nucleotide (CBASS, Pycsar) defenses (F,I); and DNA-mimicking proteins (Ocr, ArdA, Ral) inhibit type I restriction-modification systems (J).

An active Abi system senses bacteriophage infection, and upon detecting phage infection, the bacterial cell triggers self-destructive mechanisms that eventually destroy the infected bacterial cell. In Bacillus subtilis , defense-associated sirtuin 2 (DSR-2) identifies and senses phage tail tube proteins. DSR-2, which has an N-terminal sirtuin (SIR2) domain, acts as an active NADase that leads to NAD + depletion, growth arrest and eventual death of infected cells. SPβ phages evolved to encode a protein called DSR antidefense 1 (DSAD1). DSAD1 directly binds DSR2 and inhibits NAD + depletion, which allows phages to carry out their lytic life cycle. Similarly, in Bacillus subtilis , the integrative and conjugative element ICEBs1 contains the SpbK gene. SpbK provides immunity against bacteriophage infections through an abortive mechanism. SpbK is an NADase activated in the presence of the phage portal protein YonE. Active YonE prompts NAD⁺ depletion and eventually promotes growth arrest in infected cells (Figure ). The SPβ-like phage Φ3T encodes a protein similar to DSAD1 called “nip” that interferes with the NADase activity of SpbK (Figure ), thereby enabling phages to carry out their lytic lifestyle. Pectobacterium atrosepticum mediate self-protection from bacteriophage infections via the classical ToxIN-mediated Abi system. A flagellum-dependent bacteriophage of the Myoviridae family ΦTE escaped from the ToxIN system of P. atrosepticum via molecular RNA mimicry. The phage ΦTE encodes a short sequence called “Pseudo-ToxI”, similar to ToxI, which allows phages to replicate. In T4-like phages, the TifA gene prevents the ToxIN type III Abi system of P. atrosepticum. TifA forms a high-molecular-weight ribonucleoprotein complex by binding with the endonuclease ToxN, the ribonucleic acid ToxI, which halts the abortive action of the ToxIN system (Figure ). TifA forces ToxN to bind with cellular 16S rRNA, thus enabling replication of the phage. DarTG, another well-known toxin–antitoxin system, also exists in Vibrio cholerae. DarTG consists of the ADP-ribosylase DarT and the de-ADP-ribosylase DarG. DarTG is active against the lytic bacteriophage ICP1. A recent study indicated that ICP1 phages evolved to evade the DarTG-mediated defense system with AdfB, which abrogates the toxin DarT (AdfA in E. coli) ^, likely through direct interactions with the toxin–antitoxin system DarTG (Figure ). The Gabija system comprises two genes, gajA and gajB, which encode a DNA endonuclease and a UvrD-like helicase domain, respectively, and the anti-Gabija protein Gad1 blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Gad2 (Figure ) is an enzyme with a nucleotidyltransferase protein domain, suggesting that it inhibits Gabija through a mechanism of action different from that of Gad1. Another abortive defense mechanism is Thoeris. Phages evolve with Tad1 (Leavitt, Azita et al. 2022) and Tad2 to evade Thoeris defense actions. The immunological signaling molecules generated by Thoeris TIR domain proteins in response to phage infection are sequestered by the anti-Thoeris protein Tad2, which functions as a “sponge”. The Hachiman system (Cui, Y et al., 2025) is blocked by Hachiman antidefense 1 (Had1) (Yirmiya, Erez et al. 2024). CBASS and Pycsar are both abortive antiphage defense systems that promote cell death. Phages that produce Acb1 and Apyc1 disrupt CBASS and Pycsar host defense (Hobbs, Samuel J., et al. 2022). Acb1 rapidly degrades the CBASS signals 3′3′-cGAMP, cUA and cAAA but does not cleave cGG, demonstrating that Acb1 is responsible for the broad cyclic nucleotide hydrolysis activity observed in T4-infected cell lysates. Acb1 and Apyc1 block downstream effector activation and protect against CBASS and Pycsar defense in vivo. Apyc1 efficiently hydrolyses a wide range of cyclic mononucleotides (Figure ). The phage counterdefense mechanism is also capable of producing DNA-mimic proteins such as Ocr, ArdA and Ral, which inhibit some restriction modifications (R-Ms) by blocking REase. In addition, uncharacterized accessory genes (AGs) elicit programmed cell death (PCD), providing a double-layer counter defense against antiphage defense systems of host bacteria (Figure ). In addition, the loss of AGs exposes phages to the R–M system of the host bacteria.

Counterdefense Signaling against the CRISPR-Cas System and CRISPR-Cas-Supervised System

There are different types of CRISPR-Cas defense systems, and they specifically target invading nonself DNA or RNA. Anti-CRISPR proteins provide counterdefense across five CRISPR-Cas subtypes, with the exception of the type IV subtype. Pinilla Redondo, Rafael et al. reported that CRISPR-Cas counterdefense signaling is mediated by novel anti-CRISPR proteins (ACRs) of chromosomal as well as extrachromosomal mobile genetic elements (MGEs) in Enterobacteriaceae and Pseudomonas species. Li, Yuping et al. (2021) proposed a framework of “phage autonomy” that elevates and evades typical CRISPR-Cas immunity at the community level with assisted support from MGEs. However, the quick turn “ON/OFF” of the ACR/ACA operon may potentiate the evolution of novel hyperactive Acr proteins (HA-ACRs). In addition to ACR/ACA or HA-ACR, newly evolved phages evade CRISPR-Cas immunity with the help of small RNA molecules and nuclei, such as protective proteaceous barriers. For example, the promoters of several antiphage immune systems, such as the recently identified Nezha and Gabija, as well as eight yet unknown systems with unique defense domains, are targeted and inhibited by regulatory CRISPR RNA-like RNAs (crlRNAs). When viral anti-CRISPR proteins impede CRISPR-Cas, the guided immune systems cause abortive infections (Figure ), providing population-level anti-CRISPR protection.

9.

CRISPR-Cas and CRISPR-Cas-supervised defense and counter defense signaling. (A) Counterdefense by anti-CRISPR proteins (ACRs), anti-CRISPR-associated proteins (Aca), and hyperactive Acr proteins (HA-ACRs), (B) counterdefense by small noncoding RNA anti-CRISPR-associated proteins (Racrs).

Furthermore, in jumbo phages, nucleus-like proteaceous compartments (NLPCs) protect phage DNA from DNA-targeting CRISPR-Cas defense elements by providing a physical barrier. However, NLPC failed to provide immunity against type IIIA RNA-targeting defenses via the CRISPR-Cas system. Small noncoding RNA anti-CRISPR (Racrs) are a distinct type of CRISPR-Cas inhibition signal (Figure ), where Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units. Prophage-encoded Racr strongly inhibits the type I–F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex.

Counterdefense Signaling against BREX and Type-I, R–M Systems

Anti-BREX

The bacteriophage exclusion (BREX) antiphage immunity system involves widespread bacterial immunity against phages. Elements of type-I BREX recognize nonpalindromic 6-bp DNA motifs and methylate them to discriminate self-dsDNA from nonself-genetic materials. Recently, Li Xu et al. (2024) elucidated the molecular anti-BREX signaling pathway in which the T7 phage produces overcoming classical restriction (Ocr), a DNA mimic protein. Ocr inhibits the self-DNA methylation function of BrxX (Figure ) by forming a stable Ocr₂Brx₂ tetramer complex. Ocr from the bacteriophage T7 inhibits all known families of type I R-M systems by mimicking the B-form of DNA. Ocr also competitively inhibits type I DNA restriction enzymes by preventing them from binding to their DNA target and is one of the very few characterized antirestriction proteins (Walkinshaw, M. D., et al. 2002). In addition to Ocr, the BREX system is also inhibited by SAM lyase and by OrbA (Figure ) in ICP1 phages.

10.

Bacteriophage exclusion (BREX) defense system and counter defense signaling. (A) BREX counterdefense by Ocr (overcoming classical restriction), (B) BREX counterdefense by OrbA, (C) BREX counterdefense by SAM lyase.

Counterdefense Signaling against Retron, Septu and Other

Escape mutant phages acquire diverse mutations and evade diverse antiphage systems, including Septu, Retron, etc. Retron and Hachiman escape mutant phages of diverse types with mutations in the single stand binding protein (SSB) protein of the gp 2.5 family. In addition, Septu escaped mutant phages with tail fiber mutant proteins and tail fiber chaperone proteins, including proteins of unknown function. The escape mutant phages also have diverse mutant proteins linked mainly to DNA replication- and DNA maintenance-associated processes, such as DNA primase-helicase, predicted DNA polymerase-predicted SSB DNA-binding proteins (including the Gp 2.5 family and RecA family), RecBCD inhibitors, DNA second step protein A1, DNA methylase, predicted phosphatase, ribronuclease reductase, and predicted terminase, and predicted dUMP hydoxymethylase. A major phage tail protein activates the abortive antiphage systems AbiG and AbiP. Mutations in the tail protein-encoding gene allow phages to escape these defenses (Mosterd, Grafakou et al. 2025).

Other Factors Conferring Resistance to Phages

There are many distinguished mechanisms explaining the wide variety of reasons behind the emergence of PRT (Figure ) beyond mutations in phage receptors, receptor-associated proteins, and AAPD counterevolution. PRT emerges as a result of evolutionary forces that induce mutations in many essential components of bacteria, including (i) stress response systems, (ii) RNA polymerase, (iii) hypothetical/protein of unknown function, (iv) molecular chaperones, (v) DNA maintenance machineries, (vi) transcription factors, (vii) structural integrity-related genes, (viii) amino acid biosynthesis genes, (ix) nitrate respiration genes, (x) toxin biosynthesis genes, (xi) multidrug efflux components, (xii) truncated second messenger (c-di-GMP), (xiii) hypoxic conditions, (xiv) novel histidine kinase response regulators (popS, popR), and (xv) phase-variable hypermutable loci. Additionally, coevolutionary and evolutionary phage selection pressure and convergent evolution also direct the emergence of PRT (Avrani S et al.2015).

11.

Other factors linked to the emergence of phage resistance. (A) Multiple signaling mechanisms provide immunity against phage infections. (B) Multiple factors contribute to phage insensitivity or impaired phage adsorption.

High population density of lytic phages predominantly reduces host densities (Shymialevich D et al., 2022). However, Castledine M et al., 2025 demonstrated the emergence of phage resistance in Variovorax sp. due to an increase in the population density of host bacteria, a trait that persisted even after the phage went extinct (Castledine M et al., 2025). Coevolutionary and evolutionary phage selection pressure also contributed to PRT, for example, in the evolution of S. anatum phage resistance driven by mutations in the phage receptor protein btuB, with a fitness cost of reduced antibiotic resistance (Figure ). This trade-off varies with the coevolutionary dynamics stage (Zhao Y et al., 2024). The marine cyanobacterium Synechococcus WH5701 confers resistance to the Syn9 cyanophage due to adaptive loss of expression of leucine tRNA (Leu^TAA tRNA), resulting in insufficient cellular levels of Leu^TAA tRNA (Figure ). Although the R–M system and the Gabija defense system exist inside the host Synechococcus WH5701, they do not contribute to phage resistance (Zborowsky S et al., 2024). Evolutionary and coevolutionary phage training both enhance phage effectiveness against bacteria, but coevolutionary training provides superior outcomes in delaying resistance emergence and improving phage infectivity, making it a promising approach for phage therapy development (Ngiam L et al., 2024). Physiological oxygen levels also play an important role in the development of phage therapies against multidrug-resistant P. aeruginosa because hypoxia can increase the speed of phage resistance development and affect mutation patterns (Schumann A et al., 2022). Novel histidine kinase response regulators, i.e., popS and popR, control the expression of APDs, such as CRISPR-Cas and R-M signaling, and mutations in popS and popR also contribute to the emergence of PRT (Lucas-Elio P et al., 2021). Phase-variable hypermutable loci in bacteria produce herd immunity by generating resistant subpopulations that shield susceptible bacteria from bacteriophage infection, thus limiting phage spread across bacterial meta-populations (Turkington CJR et al., 2019). Plasmid-induced interference in phage–bacteria interactions also contributed to the emergence of PRT. For example, the Streptococcus lactis strain ME2 carries a 30 megadalton plasmid (pME0030 or pTR2030) that prevents efficient adsorption of phages to the bacterial cell surface. Mutants lacking this plasmid exhibit increased phage adsorption and sensitivity, indicating that this plasmid-linked mechanism blocks phage attachment (Sanders ME et al., 1984). The diversity of host genetic determinants also plays a major role in the development of PRT (Mutalik et al., 2020), such as alterations in protein expression patterns (Moreirinha C et al., 2018), SNPs in the Pf1 prophage region (Lim WS et al., 2027), and the upregulation of O-specific antigens (Spus M et al., 2015). Furthermore, Gp21, a protein expressed by vB_Pae_QDWS phages, blocks phage infection by inhibiting type IV pilus-mediated twitching motility and pilus biosynthesis in P. aeruginosa PAO1. Cyclic di-GMP (c-di-GMP) is a secondary messenger that plays a pivotal role in regulating adaptive bacterial responses. Defective c-di-GMP synthesis in the B. abortus strain Bab8416 contributed to the emergence of PRT. In L. plantarum, glgB, murG, and relA are involved in peptidoglycan synthesis in bacterial cell walls, as well as bacterial stress response mechanisms, notably under conditions of amino acid deficiency. Mutations in glgB, murG, and relA are associated with PRT. The Mycobacterium tuberculosis protein Rv2617c is associated with the stress response and provides immunity against phage infection. Staphylococcus aureus clinical isolates of spontaneous phage-resistant mutants exhibit a single missense mutation in the RNA polymerase β′ subunit (rpoC). Moreover, mutations in glycosyltransferase family 1 (GT-1) and hypothetical outer membrane protein (homP) also lead to bacterial phage resistance in Pseudomonas plecoglossicida isolated from sewage. In addition, Wang, Xuan et al. (2023) engineered a phage-resistant E. coli BL21 strain with the help of Tn5 transposons containing mutations in multiple genes, such as ecpE (fimbrial chaperone), nohD (DNA packaging protein), nrdR (linked to DNA synthesis), and livM (high-affinity branched chain amino acid transport system). In E. coli , phage resistance is associated with a point mutation (L544F) in the cell wall integrity-related gene yciM and a nonsense point mutation (Q18*) in the transcription factor rfaH. Furthermore, PRT emerges in the K. pneumoniae strain FK1979 due to a mutation in the transcription factor rfaH along with galU. Moreover, Lactobacillus lactis and Lactobacillus cremoris confer PRT due to mutations in the polysaccharide pellicle and pbp2b, a penicillin-binding protein involved in peptidoglycan biosynthesis. Genome engineering via the insertion of an alternative R–M defense system (sspBCDE) and mutant variants of fhuA and trxA also contributed to the emergence of a broad range of phage resistance in E. coli. TrxA is known to have molecular chaperone functions and to be involved in cellular redox processes. Furthermore, the phage-resistant spontaneous mutant strain P. aeruginosa PAO1-R1 has more than 60 SNPs closely linked to membrane components or the cell surface envelope, including the amino acid biosynthesis-related genes mtnC and amiE and the nitrate respiration gene napA. Recent research by Schumann, Sue et al. (2022) revealed that hypoxic conditions greatly reduced the surface adhesion properties of phages on the cell surface of P. aeruginosa. However, phage time-kill and one-step growth kinetics remain unaffected by hypoxic conditions. In bacteriophage-insensitive mutant bacteria, multiple SNPs were detected, including amn, galU, ompH, pchE, pntB, ssg and wapH. However, PRT emerges in P. aeruginosa due to large deletions in the coding regions of hmgA and galU, along with more than two hundred genes essential for pathogenicity and survival, including osmotic stress-, amino acid-, exoY toxin-, pyrroloquinoline biosynthesis- and multidrug efflux-related genes. PRT emerges in P. aeruginosa due to mutations in LPS synthesis genes along with global regulatory genes such as yqjG and vfr. In L. plantarum, mutations in major virulence-associated genes and transcriptional regulators, such as nmpC, hilA, hilD, and mig5, are associated with PRT.

Conclusion and Future Directions

Phage therapy offers excellent options against multidrug-resistant bacteria. However, recent studies have reported the ineffectiveness of phage therapy against drug-resistant bacteria from many different sectors. The emergence of PRT hinders the current process of phage therapy against emerging multidrug-resistant bacteria. This review discusses the mechanism of phage resistance development and highlights the contributing roles of fast-evolving bacteria and phages. Phage invasion is mediated by highly dynamic and specific interactions between diverse cell surface architectures of bacteria and phage surfaces or tail fiber proteins. Mutations in phage receptors offer the first layer of defense against phage infection and are the basic reason for PRT. Second, in addition to mutational events in phage receptor- or receptor-associated genes, the APD systems of the host contribute to PRT. However, rapid evolutionary or coevolutionary forces direct changes in the phage genome and attack APDs via AAPD counter defense signaling. Again, AAPD signaling direct the evolution of new APDs, which again contributed to the emergence of PRT. In essence, evolutionary or coevolutionary forces treat, train, and evolve the genomes of bacteria as well as phages, keeping APD on the one hand and AAPD on the other hand. PRT emerges between successful genomic integration of new APD or AAPD signaling modules. As such, the use of bacteria and phages as separate entities may be quite elusive and misleading in an epigenetic context. Additionally, the present narrative of the arms race between phages and bacteria is also part of such an illusion. Several epigenetic components (e.g., extracellular contractile ejection systems, MGEs) act simultaneously, participate in the entire evolutionary process of APD and AAPD and contribute to the emergence of PRT. We believe that this review may be useful for advancing phage therapy and may also direct new lines of research toward phage-bacteria signaling.

Glossary

Abbreviations

(MPR)

mutant phage receptor

(NAPD)

new antiphage defense

(APD)

antiphage defense

(AAPD)

anti-antiphage defense

(PRT)

phage resistance trait

(GNB)

gram-negative bacteria

(GPB)

gram-positive bacteria

(AFB)

acid-fast bacteria

(R-M)

restriction modification

(Abi)

abortive infection

(DISARM)

defense Island System Associated with Restriction-Modification

(EP)

drug efflux pump

(PAS)

phage–antibiotic synergy

(ADS)

antimicrobial drug sensitivity

(LPS)

lipopolysaccharide

(CPS)

capsule polysaccharide

(EPS)

exopolysaccharide

(WTA)

wall teichoic acid

(GPL)

glycopeptidolipid

(ompA)

outer membrane protein A

(SNPs)

single nucleotide polymorphisms

(galU)

glucose-1-phosphate uridylyltransferase

(PBD)

phage-borne depolymerase

(GTP)

guanosine triphosphate

(mps1)

MbtH-like protein 1

(mps2)

MbtH-like protein 2

(homP)

hypothetical outer membrane protein

(TAs)

teichoic acids

(Rha)

l-rhamnose

(UGPase)

UTP-glucose-1-phosphate uridylyltransferase

(RboP)

ribitol-phosphate

(T4P)

type-iv pili

(OMP)

outer membrane protein

(BtuB)

vitamine-B12 transporter

(T2SS)

type II secretion system

(T3SS)

type III secretion system

(TCS)

MbtH-like protein 2

(CSP)

cell surface layer protein

(SLH)

surface layer homology

(SCWPs)

secondary cell wall polysaccharides

(RBP)

receptor-binding protein

(PIAS)

phage-induced antibiotic sensitivity

(DSR-2)

defense-associated sirtuin 2

(SIR2)

N-terminal sirtuin

(DSAD1)

DSR antidefense 1

(Gad1)

anti-Gabija protein 1

(Gad2)

anti-Gabija protein 2

(Tad1)

anti-Thoeris protein 1

(Tad2)

anti-Thoeris protein 2

(Had1)

hachiman antidefense 1

(AGs)

uncharacterized accessory genes

(PCD)

programmed cell death

(ACRs)

anti-CRISPR proteins

(MGEs)

mobile genetic elements

(HA-ACRs)

hyperactive Acr proteins

(crlRNAs)

CRISPR RNA-like RNAs

(LLPCs)

nucleus-like proteaceous compartments

(Racrs)

RNA anti-CRISPR

(BREX)

bacteriophage exclusion

(Ocr)

overcoming classical restriction

(SSB)

single stand binding protein

(Leu^TAA tRNA)

leucine tRNA

(c-di-GMP)

cyclic dimeric guanosine monophosphate

(GT-1)

glycosyltransferase family 1.

The authors declare no competing financial interest.