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Updated: Jun 14, 2026

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems
Published on: October 14, 2025
Mobile regulatory cassettes mediate modular shuffling in T4-type phage genomes
Christine Arbiol1, André M Comeau, Mzia Kutateladze
1Institut d'Exploration Fonctionnelle des Génomes, CNRS-IFR109, Toulouse, France.
Abstract:
Coliphage phi1, which was isolated for phage therapy in the Republic of Georgia, is closely related to the T-like myovirus RB49. The approximately 275 open reading frames encoded by each phage have an average level of amino acid identity of 95.8%. RB49 lacks 7 phi1 genes while 10 phi1 genes are missing from RB49. Most of these unique genes encode functions without known homologs. Many of the insertion, deletion, and replacement events that distinguish the two phages are in the hyperplastic regions (HPRs) of their genomes. The HPRs are rich in both nonessential genes and small regulatory cassettes (promoter(early) stem-loops [PeSLs]) composed of strong sigma(70)-like promoters and stem-loop structures, which are effective transcription terminators. Modular shuffling mediated by recombination between PeSLs has caused much of the sequence divergence between RB49 and phi1. We show that exchanges between nearby PeSLs can also create small circular DNAs that are apparently encapsidated by the virus. Such PeSL "mini-circles" may be important vectors for horizontal gene transfer.
Insights
Coliphage phi1 and RB49 share high amino acid identity but differ in gene content, particularly within hyperplastic regions. Recombination between promoter elements drives divergence and may facilitate horizontal gene transfer via mini-circles.
Area of Science:
- Microbiology
- Virology
- Genomics
Background:
- Coliphage phi1, used in phage therapy, is closely related to the T-like myovirus RB49.
- Both phages encode approximately 275 open reading frames with 95.8% average amino acid identity.
Purpose of the Study:
- To investigate the genomic differences and evolutionary mechanisms between coliphage phi1 and RB49.
- To understand the role of hyperplastic regions and promoter elements in phage genome evolution.
Main Methods:
- Comparative genomic analysis of coliphage phi1 and RB49.
- Identification and characterization of hyperplastic regions (HPRs) and promoter(early) stem-loops (PeSLs).
Main Results:
- RB49 lacks 7 phi1 genes, while 10 phi1 genes are missing from RB49, many with unknown functions.
- Genomic divergence is concentrated in HPRs, which contain nonessential genes and PeSLs.
- Recombination between PeSLs drives modular shuffling and sequence divergence.
- PeSL exchanges can generate small circular DNAs (mini-circles) encapsidated by the virus.
Conclusions:
- Modular shuffling within HPRs, mediated by PeSLs, is a key driver of sequence divergence between phi1 and RB49.
- PeSL mini-circles represent a potential mechanism for horizontal gene transfer in these phages.
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