Phage-plasmids (P-Ps) are hybrid mobile genetic elements that transfer horizontally as temperate phages and persist vertically as plasmids. A large survey of 5,742 (5,712 in the published version) P-P genomes recovered from human, animal, plant, fungal, soil, sediment, freshwater, wastewater, and saltwater environments found that their accessory genes are mostly plasmid-like, yet anti-CRISPR systems and particular antibiotic resistance genes are enriched in P-Ps relative to both plasmids and phages. The authors explicitly leave open whether P-Ps carry functionally distinct accessory repertoires, what their aggregate contribution to the spread of accessory genes is relative to other mobile element classes in natural communities, and whether their dual replication strategy (lytic phage replication plus high-copy plasmid replication) mechanistically potentiates anti-CRISPR function. This record asks for a source-faithful resolution of that linked three-part question: establish (or refute) the functional distinctness of P-P accessory genomes under controlled comparison, quantify P-P-mediated dissemination of accessory genes relative to other mobile element classes in natural microbial communities including recombination and transmission dynamics, and experimentally test the hypothesized potentiation of anti-defence genes by high-copy-number P-P replication.

Background

Mobile genetic elements (MGEs) are DNA segments that move between and within microbial cells; the main classes are plasmids (extrachromosomal circles that replicate autonomously and can spread by conjugation), bacteriophages (viruses that infect bacteria), and integrative elements such as prophages and transposons that insert into chromosomes. Temperate phages can lysogenize, i.e., persist stably inside a host rather than immediately lysing it. Phage-plasmids (P-Ps, also called phage-plasmid-like elements, PPLEs) are a hybrid class — best known from bacteriophage P1, N15, and SSU5 — that transfer between cells as viruses during lytic infection but maintain themselves as extrachromosomal plasmids during lysogeny, exploiting plasmid replication machinery to reach high intracellular copy numbers. The accessory genome denotes genes carried by an MGE that are not required for its own replication or packaging — for example antibiotic resistance genes (ARGs), CRISPR-Cas arrays (bacterial adaptive immune systems that store spacer memories of invading DNA), anti-CRISPR (Acr) genes that phages and plasmids use to disable CRISPR-Cas immunity, virulence factors, and biosynthetic pathway cassettes. Prior genomic work established that P-Ps form numerous distinct families with conserved phage modules and variable plasmid modules (Pfeifer et al. 2021), and that P-Ps can spread ARGs through infection and lysogenic conversion (Pfeifer et al. 2022); experimental studies of model P-Ps have probed their entry mechanisms (Shan et al. 2023), and genome-level analysis shows P-Ps exchange genes more frequently with plasmids than with phages and can mediate the conversion of one element type into another (Pfeifer and Rocha 2024). Against this background, Mullet, Zhang, Pruden, and Brown (preprint 2024; published as PLOS ONE 2026, where the elements are termed phage-plasmid-like elements) mined public phage and plasmid databases with a random forest classifier, recovered 5,742 unique P-P genomes (5,712 dereplicated genomes in the published version) across disparate environments, and showed that host-associated P-Ps are enriched in defense systems and ARGs while aquatic P-Ps preferentially carry biosynthetic pathway genes. Critically, anti-CRISPR systems and specific ARGs were enriched in P-Ps relative to both plasmids and phages. The mechanistic implication the authors highlight is that plasmid-style high-copy replication could raise phage burst density, and since Acr protection is known to be density dependent (multiple Acr-bearing particles cooperate to saturate CRISPR-Cas), high-copy P-P replication could potentiate anti-CRISPR function — a hypothesis they state as untested. Their discussion, retained in substance in the published version, lists the unresolved issues that define this problem: whether P-P accessory repertoires differ functionally from plasmid repertoires beyond the few identified enrichments; the unquantified overall contribution of P-Ps to accessory-gene spread relative to other mobile element classes, including their recombination and transmission dynamics in natural communities; and the need for experimental validation of the anti-defence enrichment mechanisms. Auxiliary limitations noted by the authors — incompatibility-group typing (plasmid incompatibility groups, Inc groups, are plasmid classification units based on replication/partition incompatibility, with reference databases skewed toward Enterobacteriaceae) assignable for only a minority of P-Ps, and host prediction reaching only phylum level for 58.8% — are database constraints feeding the same core question rather than separate objectives.

Problem Statement

Determine the functional distinctness and the ecological role of phage-plasmids (P-Ps / phage-plasmid-like elements) as vehicles of accessory-gene dissemination, as left open by Mullet et al. Specifically: (1) Comparative repertoires — establish, under a controlled comparison that accounts for environment of origin and host phylogeny, whether P-P accessory genomes carry functional repertoires (defense systems including anti-CRISPR, antibiotic resistance genes, virulence factors, biosynthetic cassettes) that are distinct from those of plasmids and of phages beyond the few specific enrichments (anti-CRISPR systems; cpxA, EcoI_emrE, and CTX-M-142 resistance genes) reported in the source survey. (2) Relative contribution and dynamics — quantify the contribution of P-Ps to the spread of accessory genes in natural microbial communities relative to the other major mobile-element classes (plasmids, phages, integrative elements), including the recombination and transmission dynamics (vertical versus horizontal transfer, across-host and across-time links) by which P-P-borne accessory genes disseminate. (3) Mechanism — experimentally test the source's hypothesis that the hybrid replication strategy of P-Ps (lytic phage replication combined with plasmid-derived high-copy-number replication) potentiates anti-defence functions such as anti-CRISPR systems, e.g., by raising particle density above the cooperative threshold at which Acr proteins saturate CRISPR-Cas immunity. These three components serve one overarching objective — deciding whether P-Ps are a functionally distinct class of mobile element with a quantifiable share in horizontal transfer of accessory genes and whether their hybrid replication strategy mechanistically potentiates anti-defence function. All three components are mandatory: component (3) is resolved by an experimentally testable design and its predicted outcome; a completed wet-lab experiment is not required.

References

  1. Mullet J, Zhang L, Pruden A, Brown CL (2024) 'Phage-plasmid hybrids are found throughout diverse environments and encode niche-specific functional traits', bioRxiv preprint, doi:10.1101/2024.06.18.599647, https://doi.org/10.1101/2024.06.18.599647
  2. Mullet JI, Zhang L, Pruden A, Brown CL (2026) 'Phage-plasmid-like elements are found throughout diverse environments and encode niche-specific functional traits', PLOS ONE 21(5):e0350027, doi:10.1371/journal.pone.0350027, https://doi.org/10.1371/journal.pone.0350027
  3. Pfeifer E, Moura de Sousa JA, Touchon M, Rocha EPC (2021) 'Bacteria have numerous distinctive groups of phage-plasmids with conserved phage and variable plasmid gene repertoires', Nucleic Acids Research 49(5):2655-2673, doi:10.1093/nar/gkab064, https://doi.org/10.1093/nar/gkab064
  4. Pfeifer E, Bonnín RA, Rocha EPC (2022) 'Phage-Plasmids Spread Antibiotic Resistance Genes through Infection and Lysogenic Conversion', mBio 13(5):e01851-22, doi:10.1128/mbio.01851-22, https://doi.org/10.1128/mbio.01851-22
  5. Shan X, Szabo RE, Cordero OX (2023) 'Mutation-induced infections of phage-plasmids', Nature Communications 14:2049, doi:10.1038/s41467-023-37512-x, https://doi.org/10.1038/s41467-023-37512-x
  6. Pfeifer E, Rocha EPC (2024) 'Phage-plasmids promote recombination and emergence of phages and plasmids', Nature Communications 15:1545, doi:10.1038/s41467-024-45757-3, https://doi.org/10.1038/s41467-024-45757-3
  7. Schmidtke DT, Hickey AS, Wirbel J, Lin JD, Liachko I, Sherlock G, Bhatt AS (2025) 'The prototypic crAssphage is a linear phage-plasmid', Cell Host & Microbe 33(8):1347-1362.e5, doi:10.1016/j.chom.2025.07.004, https://doi.org/10.1016/j.chom.2025.07.004
  8. Nair S, Barker CR, Bird M, Ledda A, Collins C, Morrison R, et al. (2025) 'Large-scale genomic characterisation of phage-plasmids in clinical Enterobacteriaceae isolates', bioRxiv preprint, doi:10.1101/2025.09.12.675607, https://doi.org/10.1101/2025.09.12.675607
  9. Sankaranarayanan G, Gothandam KM (2025) 'Phage-Plasmids Are Rare in Bacteria, May Exhibit Pseudolysogeny and Lack Antibiotic Resistance Genes', bioRxiv preprint, doi:10.1101/2025.11.07.687105, https://doi.org/10.1101/2025.11.07.687105
  10. Ilchenko K, Bonnin RA, Rocha EPC, Pfeifer E (2026) 'Efficient detection and typing of phage-plasmids', mBio, doi:10.1128/mbio.03000-25, https://doi.org/10.1128/mbio.03000-25
  11. Zhang Z, Shi C (2026) 'P1-like phage-plasmids drive blaCTX-M-27 transmission through lysogenic conversion and transposition', International Journal of Antimicrobial Agents, doi:10.1016/j.ijantimicag.2025.107686, https://doi.org/10.1016/j.ijantimicag.2025.107686
  12. Wang, Xu, Lu, Yin, Chen, Zhao, Bravo, Soberón, Zheng, Sun, Peng (2025) 'Two novel trimethoprim resistance genes, dfrA50 and dfrA51, identified in phage-plasmids', Antimicrobial Agents and Chemotherapy 69:e01695-24, doi:10.1128/aac.01695-24, https://doi.org/10.1128/aac.01695-24