henhao
Sign in
MicrobiologybioRxiv
Preprint

A base-modifying enzyme extends the range of anti-phage defense provided by DNA glycosylases

Adriana Mejía-PittaiD, Zhiying ZhangiD, Amer A. HossainiD, Karolina BartosikiD, Christian Fredrick BacaiD, Christopher PeraltaiD, Henrik MolinaiD, Marianna Teplova, Sean F. BradyiD, Ronald MicuraiD, Dinshaw J. PateliD, Luciano A. MarraffiniiD

PDFDOI
Post to X
Threads on XPost to BlueskyThreads on Bluesky
In-depth full-text digest available · Jump to digest ↓

Abstract

The most abundant prokaryotic mechanisms of defense against phage predation involve the recognition and destruction of the infecting DNA. One method of counter-defense is the incorporation of modified nucleobases into the phage genome to avoid interaction with enzymes that target the viral DNA. T-even coliphages replace cytosine with 5-hydroxymethylcytosine (5hmC) that in some cases are further decorated with glucosyl groups. To explore the diversity of immunity genes that recognize 5hmC, we infected a library of metagenomic DNA inserts from uncultured, non-sequenced soil bacteria with a mutant T4 phage that harbored only non-glucosylated 5hmC on its genome. Bacteria that resisted infection carried a DNA glycosylase, Brig3, that specifically excises 5hmC nucleobases to generate abasic sites in the phage genome and prevent viral proliferation. The crystal structure of Brig3 bound to its substrate revealed a catalytic mechanism in which the 5hmC nucleobase is flipped out of the DNA into the active site and replaced by an asparagine residue that inserts into the double helix to contact the complementary guanosine. Brig3 is encoded within an operon that also encodes BapA, a hydrolase that removes glucosyl groups from glucosyl-5hmC present in the genome of otherwise Brig3-resistant T-even phages carrying this hypermodified base. Our results uncover a defense strategy in which base-modifying enzymes widen the immune response provided by DNA glycosylases.

02 /Specification & Classification

Click any tag to browse matching papers
Citations
1
OpenAlex Topics

In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (11 Oct 2026) · CC BY 4.0

Brig3, a DNA glycosylase from a soil metagenome, excises 5-hydroxymethylcytosine from T-even phage DNA; the glucose hydrolase BapA extends defense to glucosylated genomes such as wild-type T4.

T-even phages modify their cytosines (5hmC, then glucosylated 5hmC) to escape host defenses, and the earlier glycosylase Brig1 acts only on α-glucosyl-5hmC. Brig3 targets unmodified 5hmC, and its operon partner BapA strips glucose so that fully glucosylated genomes also become Brig3 substrates.

Key results

  1. ShownBrig3 alone defends E. coli against a T4 phage carrying only unmodified 5hmC, T4(5hmC), but not against wild-type T4 or a cytosine-only T4. It lowers viral DNA accumulation, and no escape plaques were seen.

    The screened clone cut T4(5hmC) PFU by at least six orders of magnitude and left T5 unaffected. qPCR at 2, 4 and 8 min after infection showed lower T4(5hmC) DNA with Brig3 than with vector. Brig3 did not protect against T4(wt) even at MOI 0.01 to 1. · Brig3 limits the replication of a T4 phage…

  2. ShownBrig3 protects against phages that have only a fraction of unmodified 5hmC, including T2, T6 and eleven BASEL T-even phages, but not non-Tevenvirinae phages. It is weak against a fully glucosylated T4 mutant.

    Plaquing was strongly reduced for T2, T6, T4(Δb-gt) and all eleven BASEL phages Bas35 to Bas45. T4(Δa-gt), with no free 5hmC, lost less than 10-fold PFU, even with overexpression. · Brig3 provides immunity against diverse phages…

  3. ShownBrig3 is a 5hmC-specific DNA glycosylase that leaves abasic sites in single- and double-stranded oligonucleotides, and degrades 5hmC-containing phage genomic DNA, most likely via multiple abasic sites. It has residual activity on uracil only.

    On a 60-nt oligonucleotide, 10 nM Brig3 acted on 5hmC, whereas uracil needed at least 1 µM; C, 5mC and T were not excised. Mass spectrometry showed an AP-site mass shift. T4(5hmC) DNA was degraded at low enzyme concentrations, while T4(wt) and plasmid DNA stayed intact. · Brig3 excises 5hmC nucleobases to generate…

  4. ShownCrystal structures show base flipping into an aromatic pocket, with N225 filling the gap in the duplex. Alanine mutants of key residues abolish or reduce defense.

    Structures at 3.0 Å (apo), 1.7 Å (product complex) and 1.6 Å (D127N substrate complex). R197A and N225A raised T4(5hmC) PFU by six and five orders of magnitude. W100A, E55A and N50A abrogated defense, and Y97A raised PFU by two orders. · An asparagine residue displaces 5hmC into the…

  5. ShownBapA, a glucose hydrolase, releases glucose from α- and β-glucosyl-5hmC in DNA, preferring α, but not from gentiobiosyl-5hmC. Together with Brig3 it extends defense to wild-type T4. D101 is required.

    Brig3 cleaved glucosylated oligonucleotides only after BapA treatment. BapA released glucose from T4(wt) DNA but not from T4(5hmC) DNA or T6 DNA. Overexpressed Brig3 plus BapA gave no detectable T4(Δa-gt) plaques. The D101N mutation drastically reduced activity in the glucose assay (Fig. 6C), Brig3 cleavage (Fig. 6D) and phage defense (Fig. 6E). · BapA removes glucose residues from α- and…; An aspartic acid is required for BapA’s activity

Methods, data and reproducibility

Functional selection of a soil metagenomic cosmid library in E. coli with a glucosyltransferase-deficient T4 phage, then subcloning, plaque and growth assays, and in vitro oligonucleotide and phage-DNA assays. X-ray crystallography, mutagenesis and an AlphaFold 3 model of BapA were used for mechanism.

  • Arizona soil metagenomic cosmid library AZ52 (about 40 kb inserts) screened against T4(5hmC) · uncultured soil microorganisms, expressed in E. coli EC100 · n = approx. 20 million clones
  • Cosmid insert carrying the brig3-bapA defense island, sequenced · unknown soil bacterium · n = 35.4 kb insert · GenBank PX942695
  • Phage panel: T4 variants, T2, T6 and BASEL collection phages Bas35 to Bas45 plus non-Tevenvirinae controls · E. coli phages · n = eleven BASEL T-even phages
  • X-ray structures of Brig3 apo, Brig3 with 5hmC-dsDNA, and Brig3 D127N with 5hmC-dsDNA · n = 3 structures (3.0, 1.7, 1.6 Å)
Methods & tools
AlphaFold 2, AlphaFold 3, HHpred, DALI, PADLOC, PHASER, PHENIX, Coot, UniDec, Glucose-Glo assay, PyMOL, UCSF ChimeraX
Reagents & environment
UniDec GUI v.1.0.10 for mass spectra deconvolution; no other software environment is stated.
Reproducibility gaps
Strains, plasmids, phages and oligonucleotides are listed only in a Supplementary Data File. No PDB accession or code repository is printed. A provisional patent has been filed.
Limitations (4)
  • The organism that originally carried the defense island cannot be determined, because the insert had almost no match in GenBank. · stated by the authors
  • A crystal structure of BapA could not be obtained, so its mechanism rests on an AlphaFold 3 model. The Brig1 comparison also uses a model, and the authors call their data limited. · stated by the authors
  • All defense data come from a laboratory E. coli K-12-derived host with plasmid or cosmid expression, and mostly from T4 relatives. · noted in this summary
  • BapA's role in vivo is inferred from indirect assays; its glucose-release signal on some genomes may be saturated, and passaging phage through BapA alone did not change sensitivity to Brig3. · noted in this summary

Who should read:Read this if you study bacterial anti-phage defense, DNA glycosylase mechanism, or how phages evade defenses through hypermodified bases.

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISGetz '25Pyle '24Ramirez-Cha '26Lee '26Brandt '24Wang '23
79% semantic matchMicrobiology
Landon J. Getz, Amy L. Qian, Yundan Liu+5 more·bioRxiv·Oct 29, 2025· 3 citations