Phosphorothioate DNA modification by BREX type 4 systems in the human gut microbiome

Yifeng Yuan¹, Michael S DeMott^{1

2}, Shane R Byrne^{1

3}, Katia Flores⁴, Mathilde Poyet^{1

5

6}, Mathieu Groussin^{1

6

7}, Brittany Berdy^{8

9}, John Rusine Bahunde^{6

10

11

12}, Catherine Girard^{6

13

14}, Jenni Lehtimäki^{6

15}, Audax Z P Mabulla^{6

16}, Ivan Emil Mwikarago^{6

10

17}, Yvonne Ayerki Nartey^{6

18}, Le Thanh Tu Nguyen^{1

6}, Charles A Onyekwere^{6

19}, Lewis R Roberts^{6

20}, B Jesse Shapiro^{6

21

22

23}, Tommi Vatanen^{6

8

24

25

26

27}, Laurie E Comstock⁴, Eric J Alm^{1

6

8

28

29}, Peter C Dedon^{30

31

32}

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
² Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Codomax Inc., Worcester, MA, USA.
⁴ Department of Microbiology, Duchossois Family Institute, University of Chicago, Chicago, IL, USA.
⁵ Institute of Experimental Medicine, Kiel University, Kiel, Germany.
⁶ Global Microbiome Conservancy, Kiel University, Kiel, Germany.
⁷ Institute of Clinical and Molecular Biology, Kiel University, Kiel, Germany.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁹ MGH Institute of Health Professions, Boston, MA, USA.
¹⁰ Department of Laboratory Sciences, University of Rwanda College of Medicine and Health Sciences, Kigali, Rwanda.
¹¹ United States Pharmacopeia, Rockville, MD, USA.
¹² Community Options, Derwood, MD, USA.
¹³ University of Quebec at Chicoutimi, Saguenay, QC, Canada.
¹⁴ University of Laval, Quebec, QC, Canada.
¹⁵ Finnish Environment Institute, Helsinki, Finland.
¹⁶ University of Dar es Salaam, Dar es Salaam Region, Tanzania.
¹⁷ Rwanda Food and Drug Authority, Department of Drugs and Device Registration, Division of Human Medicine and Device Registration, Kigali, Rwanda.
¹⁸ Department of Internal Medicine. School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana.
¹⁹ Department of Medicine, Lagos State University College of Medicine, Lagos, Nigeria.
²⁰ Mayo Clinic College of Medicine and Science, Rochester, MN, USA.
²¹ Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
²² McGill Genome Centre, McGill University, Montreal, QC, Canada.
²³ McGill Centre for Microbiome Research, Montreal, QC, Canada.
²⁴ Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
²⁵ Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland.
²⁶ Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
²⁷ Liggins Institute, University of Auckland, Auckland, New Zealand.
²⁸ Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, MA, USA.
²⁹ Singapore-MIT Alliance for Research and Technology, Singapore, Singapore.
³⁰ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. pcdedon@mit.edu.
³¹ Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, MA, USA. pcdedon@mit.edu.
³² Singapore-MIT Alliance for Research and Technology, Singapore, Singapore. pcdedon@mit.edu.

PMID: 41571653
PMCID: PMC12914056
DOI: 10.1038/s41467-026-68412-5

Phosphorothioate DNA modification by BREX type 4 systems in the human gut microbiome

Yifeng Yuan et al. Nat Commun. 2026.

. 2026 Jan 22;17(1):1717.

doi: 10.1038/s41467-026-68412-5.

Authors

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
² Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, MA, USA.
³ Codomax Inc., Worcester, MA, USA.
⁴ Department of Microbiology, Duchossois Family Institute, University of Chicago, Chicago, IL, USA.
⁵ Institute of Experimental Medicine, Kiel University, Kiel, Germany.
⁶ Global Microbiome Conservancy, Kiel University, Kiel, Germany.
⁷ Institute of Clinical and Molecular Biology, Kiel University, Kiel, Germany.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁹ MGH Institute of Health Professions, Boston, MA, USA.
¹⁰ Department of Laboratory Sciences, University of Rwanda College of Medicine and Health Sciences, Kigali, Rwanda.
¹¹ United States Pharmacopeia, Rockville, MD, USA.
¹² Community Options, Derwood, MD, USA.
¹³ University of Quebec at Chicoutimi, Saguenay, QC, Canada.
¹⁴ University of Laval, Quebec, QC, Canada.
¹⁵ Finnish Environment Institute, Helsinki, Finland.
¹⁶ University of Dar es Salaam, Dar es Salaam Region, Tanzania.
¹⁷ Rwanda Food and Drug Authority, Department of Drugs and Device Registration, Division of Human Medicine and Device Registration, Kigali, Rwanda.
¹⁸ Department of Internal Medicine. School of Medical Sciences, University of Cape Coast, Cape Coast, Ghana.
¹⁹ Department of Medicine, Lagos State University College of Medicine, Lagos, Nigeria.
²⁰ Mayo Clinic College of Medicine and Science, Rochester, MN, USA.
²¹ Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
²² McGill Genome Centre, McGill University, Montreal, QC, Canada.
²³ McGill Centre for Microbiome Research, Montreal, QC, Canada.
²⁴ Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
²⁵ Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Helsinki, Finland.
²⁶ Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
²⁷ Liggins Institute, University of Auckland, Auckland, New Zealand.
²⁸ Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, MA, USA.
²⁹ Singapore-MIT Alliance for Research and Technology, Singapore, Singapore.
³⁰ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. pcdedon@mit.edu.
³¹ Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, MA, USA. pcdedon@mit.edu.
³² Singapore-MIT Alliance for Research and Technology, Singapore, Singapore. pcdedon@mit.edu.

PMID: 41571653
PMCID: PMC12914056
DOI: 10.1038/s41467-026-68412-5

Abstract

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis revealed several previously undescribed PT synthesis systems, including type 4 Bacteriophage Exclusion (BREX) type 4 brx genes, which we genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd, ssp, and brx genes revealed 8 PT dinucleotide settings confirmed in 10 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

**Fig. 1. Microbial phosphorothioate (PT) DNA modifications.**
A Sulfur replaces a non-bridging phosphate oxygen in the DNA backbone in PT modifications. A limit nuclease digest of PT-containing DNA leaves PT-linked dinucleotides that can be identified and quantified by LC–MS. B Synthesis of PTs generally follows the biochemical steps performed by Dnd proteins.

**Fig. 2. Gene neighborhoods analyses based on sequence similarity networks (SSNs) of DndC and SspD proteins essential for PT synthesis.**
Sequence similarity networks (SSN) analysis was performed for the 3120 closest homologues of DndC (A) and 2132 homologues of SspD (B) in the UniProt database. Each node (circle) in the network represents one DndC or SspD protein. An edge is drawn between two nodes that have a BLAST E-value cutoff of ≤10⁻¹⁰⁰ (alignment score of 100) in the DndC SSN or 10⁻⁵⁰ (alignment score of 50) in the SspD SSN. Some nodes are filled with colors according to their genome neighborhood structure, with a representative species shown and indicated in the SSNs with a two-letter label. The node outlines are colored red when *dndD* is present in the genome neighborhood of *dndC*, and similarly for *sspD* when *sspBC* are present in the neighborhood. For better visualization, single nodes and clusters with only a few nodes were hidden. HEPN higher eukaryotes and prokaryotes nucleotide-binding, MTase methyltransferase, NTPase nucleoside triphosphatase, NTP transf, nucleotidyltransferase, top topoisomerase.

**Fig. 3. Homology analysis of BREX type 4 systems and evidence of PT synthesis.**
A The genomic organization of the BREX type 4 gene systems in *Bacteroides salyersiae* and putative *Butyricimonas faecalis*. *Vibrio cyclitrophicus* FF75 is included to demonstrate a typical *ssp* gene system and *Escherichia coli* HS to demonstrate a typical BREX type 1 system. Protein domains are color-coded and labeled. B The levels of PT dinucleotides in engineered *B. salyersiae* and *Bacteroides thetaiotaomicron* strains. Δ*brxC*, Δ*brxC brxC*_Bf, Δ*brxC sspC*_Bo, vector, and *brxC*_Bs all differ significantly from wild-type (WT) based on a two-tailed Student’s t-test with three biological replicates (p < 0.05). Data are presented as mean values ± SD. Bs: *B. salyersiae*, Bf: *Butyricimonas faecalis*, Bo: *Bacteroides ovatus*. Statistical data are provided in Supplementary Data 15 and 16.

**Fig. 4. Phylogenetic distribution of *dndCD, sspBCD*, and *brxPC* genes in human gut microbiome genomes.**
The reference phylogeny was reconstructed from the concatenated alignment of 10 ribosomal proteins. The colors of the triangles in the tree show the taxa at the order level. The occurrence of gene clusters was quantitatively presented by the colors and size of circles. For better visualization, orders containing <5 genomes were hidden. Source data are provided in Supplementary Table S5.

**Fig. 5. PT dinucleotides and PT consensus sequences in human gut microbiome isolates.**
The numbers of isolates analyzed are listed on the left. The circles represent PT dinucleotides quantified by LC–MS (left) and the presence of corresponding genes (right). The PT-modified consensus motifs were characterized in one representative isolate from each group using PT-seq. t.b.d., to be determined. *The C_PSAG motif was characterized using metagenomics PT-seq in another ongoing study. **G_PSA and G_PST were detected using LC–MS QQQ, but the exact mass was not verified by high-resolution mass spectrometry.

**Fig. 6. Biogeographical maps of PTs in bacteria with BREX type 4 systems.**
A Pie charts depict the number of single- or bi-stranded PT-modified consensus sequences. The structural diagrams depict these motifs. B Analysis of the distribution of PT consensus sequences and modified sites in 1 kb upstream and downstream regions in *B. salyersiae* (**left**) and putative *B. faecalis* (**right**). The number of total motif sites in the sense strand (**upper**) or both strands (**lower**) is represented in pink. The PT-modified sites are represented in purple. The fraction of PT-modified motif sites is represented by the black line.

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Update of

Phosphorothioate DNA modification by BREX Type 4 systems in the human gut microbiome.
Yuan Y, DeMott MS, Byrne SR, Flores K, Poyet M, Groussin M, Microbiome Conservancy G, Berdy B, Comstock L, Alm EJ, Dedon PC. Yuan Y, et al. bioRxiv [Preprint]. 2024 Jun 3:2024.06.03.597175. doi: 10.1101/2024.06.03.597175. bioRxiv. 2024. Update in: Nat Commun. 2026 Jan 22;17(1):1717. doi: 10.1038/s41467-026-68412-5. PMID: 38895356 Free PMC article. Updated. Preprint.

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Phosphorothioate DNA modification by BREX type 4 systems in the human gut microbiome

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Phosphorothioate DNA modification by BREX type 4 systems in the human gut microbiome

Authors

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Conflict of interest statement

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