Phage Defense Systems Research Articles

Prophages in Vibrio.

Although tailed bacteriophages (phages) of the class Caudoviricetes are thought to constitute the most abundant and ecologically relevant group of phages that can integrate their genome into the host chromosome, it is becoming increasingly clear that other prophages are widespread. Here, we show that prophages derived from filamentous and tailless phages with genome sizes below 16 kb make up the majority of prophages in marine bacteria of the genus Vibrio. To estimate prophage prevalence unaffected by database biases, we combined comparative genomics and chemical induction of 58 diverse Vibrio cyclitrophicus isolates, resulting in 107 well-curated prophages. Complemented with computationally predicted prophages, we obtained 1,158 prophages from 931 naturally co-existing strains of the family Vibrionaceae. Prophages resembling tailless and filamentous phages predominated, accounting for 80% of all prophages in V. cyclitrophicus and 60% across the Vibrionaceae. In our experimental model, prophages of all three viral realms actively replicated upon induction indicating their ability to transfer to new hosts. Indeed, prophages were rapidly gained and lost, as suggested by variable prophage content between closely related V. cyclitrophicus. Prophages related to filamentous and tailless phages were integrated into only three genomic locations and restored the function of their integration site. Despite their small size, they contained highly diverse accessory genes that may contribute to host fitness, such as phage defense systems. We propose that, like their well-studied tailed equivalent, tailless and filamentous temperate phages are active and highly abundant drivers of host ecology and evolution in marine Vibrio, which have been largely overlooked.

The vibriophage-encoded inhibitor OrbA abrogates BREX-mediated defense through the ATPase BrxC.

Bacteria and phages are locked in a co-evolutionary arms race where each entity evolves mechanisms to restrict the proliferation of the other. Phage-encoded defense inhibitors have proven powerful tools to interrogate how defense systems function. A relatively common defense system is BREX (bacteriophage exclusion); however, how BREX functions to restrict phage infection remains poorly understood. A BREX system encoded by the sulfamethoxazole and trimethoprim (SXT) integrative and conjugative element, VchInd5, was recently identified in Vibrio cholerae, the causative agent of the diarrheal disease cholera. The lytic phage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) that co-circulates with V. cholerae encodes the BREX-inhibitor OrbA, but how OrbA inhibits BREX is unclear. Here, we determine that OrbA inhibits BREX using a unique mechanism from known BREX inhibitors by directly binding to the BREX component BrxC. BrxC has a functional ATPase domain that, when mutated, not only disrupts BrxC function but also alters how BrxC multimerizes. Furthermore, we find that OrbA binding disrupts BrxC-BrxC interactions. We determine that OrbA cannot bind BrxC encoded by the distantly related BREX system encoded by the aSXT VchBan9, and thus fails to inhibit this BREX system that also circulates in epidemic V. cholerae. Lastly, we find that homologs of the VchInd5 BrxC are more diverse than the homologs of the VchBan9 BrxC. These data provide new insight into the function of the BrxC ATPase and highlight how phage-encoded inhibitors can disrupt phage defense systems using different mechanisms.IMPORTANCEWith renewed interest in phage therapy to combat antibiotic-resistant pathogens, understanding the mechanisms bacteria use to defend themselves against phages and the counter-strategies phages evolve to inhibit defenses is paramount. Bacteriophage exclusion (BREX) is a common defense system with few known inhibitors. Here, we probe how the vibriophage-encoded inhibitor OrbA inhibits the BREX system of Vibrio cholerae, the causative agent of the diarrheal disease cholera. By interrogating OrbA function, we have begun to understand the importance and function of a BREX component. Our results demonstrate the importance of identifying inhibitors against defense systems, as they are powerful tools for dissecting defense activity and can inform strategies to increase the efficacy of some phage therapies.

A Vibrio cholerae Type IV restriction system targets glucosylated 5-hydroxymethylcytosine to protect against phage infection.

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has acquired and/or evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized that more were encoded in V. cholerae given the low diversity of phages that have been isolated, which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16-kB region of the Vibrio pathogenicity island-2, which we name TgvA and TgvB (Type I-embedded gmrSD-like system of VPI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modifications are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff, becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We also show that the Type I restriction-modification system that embeds the tgvAB genes protects against phage T3, secΦ18, secΦ27, and λ, suggesting that this region is a phage defense island. Our study uncovers a novel Type IV restriction system in V. cholerae, increasing our understanding of the evolution and ecology of V. cholerae, while highlighting the evolutionary interplay between restriction systems and phage genome modification.IMPORTANCEBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. In this study, we discover a Type IV restriction system encoded in V. cholerae, which we name TgvAB, and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation.

Bacterial homologs of innate eukaryotic antiviral defenses with anti-phage activity highlight shared evolutionary roots of viral defenses

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes.

Phages carry orphan antitoxin-like enzymes to neutralize the DarTG1 toxin-antitoxin defense system.

The astounding number of anti-phage defenses encoded by bacteria is countered by an elaborate set of phage counter-defenses, though their evolutionary origins are often unknown. Here, we discover an orphan antitoxin counter-defense element in T4-like phages that can overcome the bacterial toxin-antitoxin phage defense system, DarTG1. The DarT1 toxin, an ADP-ribosyltransferase, modifies phage DNA to prevent replication while its cognate antitoxin, DarG1, is an ADP-ribosylglycohydrolase that reverses these modifications in uninfected bacteria. The orphan phage DarG1-like protein, which we term anti-DarT factor NADAR (AdfN), removes ADP-ribose modifications from phage DNA during infection thereby enabling replication in DarTG1-containing bacteria. AdfN, like DarG1, is in the NADAR superfamily of ADP-ribosylglycohydrolases found across domains of life. We find divergent NADAR proteins in unrelated phages that likewise exhibit anti-DarTG1 activity, underscoring the importance of ADP-ribosylation in bacterial-phage interactions, and revealing the function of a substantial subset of the NADAR superfamily.

Structural and functional characterization of cyclic pyrimidine-regulated anti-phage system

3’,5’-cyclic uridine monophosphate (cUMP) and 3’,5’-cyclic cytidine monophosphate (cCMP) have been established as bacterial second messengers in the phage defense system, named pyrimidine cyclase system for anti-phage resistance (Pycsar). This system consists of a pyrimidine cyclase and a cyclic pyrimidine receptor protein. However, the molecular mechanism underlying cyclic pyrimidine synthesis and recognition remains unclear. Herein, we determine the crystal structures of a uridylate cyclase and a cytidylate cyclase, revealing the conserved residues for cUMP and cCMP production, respectively. In addition, a distinct zinc-finger motif of the uridylate cyclase is identified to confer substantial resistance against phage infections. Furthermore, structural characterization of cUMP receptor protein PycTIR provides clear picture of specific cUMP recognition and identifies a conserved N-terminal extension that mediates PycTIR oligomerization and activation. Overall, our results contribute to the understanding of cyclic pyrimidine-mediated bacterial defense.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.

Structure-functional characterization of Lactococcus AbiA phage defense system.

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

Strategic combination of bacteriophages with highly susceptible cells for enhanced intestinal settlement and resistant cell killing

Avian pathogenic Escherichia coli (APEC) causes enormous economic losses and is a primary contributor to the emergence of multidrug resistance (MDR)-related problems in the poultry industry. Bacteriophage (phage) therapy has been successful in controlling MDR, but phage-resistant variants have rapidly emerged through the horizontal transmission of diverse phage defense systems carried on mobile genetic elements. Consequently, while multiple phage cocktails are recommended for phage therapy, there is a growing need to explore simpler and more cost-effective phage treatment alternatives. In this study, we characterized two novel O78-specific APEC phages, φWAO78-1 and φHAO78-1, in terms of their morphology, genome, physicochemical stability and growth kinetics. Additionally, we assessed the susceptibility of thirty-two O78 APEC strains to these phages. We analyzed the roles of highly susceptible cells in intestinal settlement and fecal shedding (susceptible cell-assisted intestinal settlement and shedding, SAIS) of phages in chickens via coinoculation with phages. Furthermore, we evaluated a new strategy, susceptible cell-assisted resistant cell killing (SARK), by comparing phage susceptibility between resistant cells alone and a mixture of resistant and highly susceptible cells in vitro. As expected, high proportions of O78 APEC strains had already acquired multiple phage defense systems, exhibiting considerable resistance to φWAO78-1 and φHAO78-1. Coinoculation of highly susceptible cells with phages prolonged phage shedding in feces, and the coexistence of susceptible cells markedly increased the phage susceptibility of resistant cells. Therefore, the SAIS and SARK strategies were demonstrated to be promising both in vivo and in vitro.

Accumulation of defense systems in phage-resistant strains of Pseudomonas aeruginosa.

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.

Diverse physiological roles of the MqsR/MqsA toxin/antitoxin system

Abstract There is tremendous interest in the use of bacteriophages (phages) to combat multi-drug resistant bacteria. However, to implement successfully phage therapy, host defense systems must be understood. Toxin/antitoxins (TAs) are the most prevalent phage defense system, and the MqsR/MqsA TA system is one of the best-studied systems. This phage-defense system was discovered in a whole-cell, population-averaged, transcriptome study designed to elucidate the biofilm-related genes of Escherichia coli in 2004. Biofilms are cells cemented to themselves or to surfaces. Since its characterization (as of April 2024), MqsR/MqsA has been utilized in over 1200 manuscripts, although its role in cell physiology has been contested. Here, we summarize the important physiological roles of this TA system, including its role in (i) the general stress response via repression of rpoS, (ii) biofilm formation via repression of csgA, (iii) combating bile acid stress in the gastrointestinal tract by inhibiting uptake of the bile salt deoxycholate, (iv) oxidative stress based on single-cell transcriptome studies, and (v) phage defense leading to the persister state.

Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage.

To date, there are no reports of phage infection-inducing persistence. Therefore, our results are important since we show for the first time that a phage-defense system, the MqsRAC toxin/antitoxin system, allows the host to survive infection by forming persister cells, rather than inducing cell suicide. Moreover, we demonstrate that the MqsRAC system works in concert with restriction/modification systems. These results imply that if phage therapy is to be successful, anti-persister compounds need to be administered along with phages.

Phage selection drives resistance-virulence trade-offs in Ralstonia solanacearum plant-pathogenic bacterium irrespective of the growth temperature.

While temperature has been shown to affect the survival and growth of bacteria and their phage parasites, it is unclear if trade-offs between phage resistance and other bacterial traits depend on the temperature. Here, we experimentally compared the evolution of phage resistance-virulence trade-offs and underlying molecular mechanisms in phytopathogenic Ralstonia solanacearum bacterium at 25 °C and 35 °C temperature environments. We found that while phages reduced R. solanacearum densities relatively more at 25 °C, no difference in the final level of phage resistance was observed between temperature treatments. Instead, small colony variants (SCVs) with increased growth rate and mutations in the quorum-sensing (QS) signaling receptor gene, phcS, evolved in both temperature treatments. Interestingly, SCVs were also phage-resistant and reached higher frequencies in the presence of phages. Evolving phage resistance was costly, resulting in reduced carrying capacity, biofilm formation, and virulence in planta, possibly due to loss of QS-mediated expression of key virulence genes. We also observed mucoid phage-resistant colonies that showed loss of virulence and reduced twitching motility likely due to parallel mutations in prepilin peptidase gene, pilD. Moreover, phage-resistant SCVs from 35 °C-phage treatment had parallel mutations in type II secretion system (T2SS) genes (gspE and gspF). Adsorption assays confirmed the role of pilD as a phage receptor, while no loss of adsorption was found with phcS or T2SS mutants, indicative of other downstream phage resistance mechanisms. Additional transcriptomic analysis revealed upregulation of CBASS and type I restriction-modification phage defense systems in response to phage exposure, which coincided with reduced expression of motility and virulence-associated genes, including pilD and type II and III secretion systems. Together, these results suggest that while phage resistance-virulence trade-offs are not affected by the growth temperature, they could be mediated through both pre- and postinfection phage resistance mechanisms.

Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system.

Although numerous phage defense systems have recently been discovered in bacteria, how these systems defend against phage propagation or sense phage infections remains unclear. The Escherichia coli AbpAB defense system targets several lytic and lysogenic phages harboring DNA genomes. A phage-encoded single-stranded DNA-binding protein, Gp32, activates this system similar to other phage defense systems such as Retron-Eco8, Hachiman, ShosTA, Nhi, and Hna. DNA replication inhibitors or defects in DNA repair factors activate the AbpAB system, even without phage infection. This is one of the few examples of activating phage defense systems without phage infection or proteins. The AbpAB defense system may be activated by sensing specific DNA-protein complexes.

Adaptation to bile and anaerobicity limits Vibrio cholerae phage adsorption.

Vibrio cholerae is the bacterial pathogen responsible for cholera, a diarrheal disease that impacts people in areas without access to potable water. In regions that lack such infrastructure, cholera represents a large proportion of disease outbreaks. Bacteriophages (phages, viruses that infect bacteria) have recently been examined as potential therapeutic and prophylactic agents to treat and prevent bacterial disease outbreaks like cholera due to their specificity and stability. This work examines the interaction between V. cholerae and vibriophages in consideration for a cholera prophylaxis regimen (M. Yen, L. S. Cairns, and A. Camilli, Nat Commun 8:14187, 2017, https://doi.org/10.1038/ncomms14187) in the context of stimuli found in the intestinal environment. We discover that common signals in the intestinal environment induce cell surface modifications in V. cholerae that also restrict some phages from binding and initiating infection. These findings could impact considerations for the design of phage-based treatments, as phage infection appears to be limited by bacterial adaptations to the intestinal environment.

Genomic insights into the phage-defense systems of Stenotrophomonas maltophilia clinical isolates

Stenotrophomonas maltophilia is a rapidly evolving multidrug-resistant opportunistic pathogen that can cause serious infections in immunocompromised patients. Although phage therapy is one of promising strategies for dealing with MDR bacteria, the main challenges of phage therapeutics include accumulation of phage resistant mutations and acquisition of the phage defense systems. To systematically evaluate the impact of (pro)phages in shaping genetic and evolutionary diversity of S. maltophilia, we collected 166 S. maltophilia isolates from three hospitals in southern China to analyze its pangenome, virulence factors, prophage regions, and anit-viral immune systems. Pangenome analysis indicated that there are 1328 saturated core genes and 26961 unsaturated accessory genes in the pangenome, suggesting existence of highly variable parts of S. maltophilia genome. The presence of genes in relation to T3SS and T6SS mechanisms suggests the great potential to secrete toxins by the S. maltophilia population, which is contrary to the conventional notion of low-virulence of S. maltophilia. Additionally, we characterized the pan-immune system maps of these clinical isolates against phage infections and revealed the co-harboring of CBASS and anti-CBASS in some strains, suggesting a never-ending arms race and the co-evolutionary dynamic between bacteria and phages. Furthermore, our study predicted 310 prophage regions in S. maltophilia with high genetic diversity. Six viral defense systems were found to be located at specific position of the S. maltophilia prophage genomes, indicating potential evolution of certain site/region similar to bacterial ‘defense islands’ in prophage. Our study provides novel insights into the S. maltophilia pangenome in relation to phage-defense mechanisms, which extends our understanding of bacterial-phage interactions and might guide the application of phage therapy in combating S. maltophilia infections.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

Phage-Defense Systems Are Unlikely to Cause Cell Suicide.

As new phage-defense systems (PDs) are discovered, the overlap between their mechanisms and those of toxin/antitoxin systems (TAs) is becoming clear in that both use similar means to reduce cellular metabolism; for example, both systems have members that deplete energetic compounds (e.g., NAD+, ATP) and deplete nucleic acids, and both have members that inflict membrane damage. Moreover, both TAs and PDs are similar in that rather than altruistically killing the host to limit phage propagation (commonly known as abortive infection), both reduce host metabolism since phages propagate less in slow-growing cells, and slow growth facilitates the interaction of multiple phage-defense systems.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin–antitoxin and related phage defense systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Comparative genomic insights into habitat adaptation of coral-associated Prosthecochloris.

Green sulfur bacteria (GSB) are a distinct group of anoxygenic phototrophic bacteria that are found in many ecological niches. Prosthecochloris, a marine representative genus of GSB, was found to be dominant in some coral skeletons. However, how coral-associated Prosthecochloris (CAP) adapts to diurnal changing microenvironments in coral skeletons is still poorly understood. In this study, three Prosthecochloris genomes were obtained through enrichment culture from the skeleton of the stony coral Galaxea fascicularis. These divergent three genomes belonged to Prosthecochloris marina and two genomes were circular. Comparative genomic analysis showed that between the CAP and non-CAP clades, CAP genomes possess specialized metabolic capacities (CO oxidation, CO2 hydration and sulfur oxidation), gas vesicles (vertical migration in coral skeletons), and cbb 3-type cytochrome c oxidases (oxygen tolerance and gene regulation) to adapt to the microenvironments of coral skeletons. Within the CAP clade, variable polysaccharide synthesis gene clusters and phage defense systems may endow bacteria with differential cell surface structures and phage susceptibility, driving strain-level evolution. Furthermore, mobile genetic elements (MGEs) or evidence of horizontal gene transfer (HGT) were found in most of the genomic loci containing the above genes, suggesting that MGEs play an important role in the evolutionary diversification between CAP and non-CAP strains and within CAP clade strains. Our results provide insight into the adaptive strategy and population evolution of endolithic Prosthecochloris strains in coral skeletons.