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[Preprint]. 2024 Jun 3:2024.06.03.597175.

doi: 10.1101/2024.06.03.597175.

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

Yifeng Yuan¹, Michael S DeMott^{1

2}, Shane R Byrne¹, Katia Flores³, Mathilde Poyet^{1

4

5}, Mathieu Groussin^{1

6

5}, Global Microbiome Conservancy^{5

7}, Brittany Berdy⁸, Laurie Comstock³, Eric J Alm^{1

8

7

9}, Peter C Dedon^{1

2

9}

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
² Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
³ Department of Microbiology, Duchossois Family Institute, University of Chicago, Chicago, IL, USA.
⁴ Institute of Experimental Medicine, Kiel University, Germany.
⁵ Global Microbiome Conservancy (https://microbiomeconservancy.org/), Kiel University, Germany.
⁶ Institute of Clinical and Molecular Biology, Kiel University, Germany.
⁷ Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, MA.
⁸ Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
⁹ Singapore-MIT Alliance for Research and Technology, Singapore.

PMID: 38895356
PMCID: PMC11185695
DOI: 10.1101/2024.06.03.597175

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

Yifeng Yuan et al. bioRxiv. 2024.

[Preprint]. 2024 Jun 3:2024.06.03.597175.

doi: 10.1101/2024.06.03.597175.

Authors

Affiliations

¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
² Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
³ Department of Microbiology, Duchossois Family Institute, University of Chicago, Chicago, IL, USA.
⁴ Institute of Experimental Medicine, Kiel University, Germany.
⁵ Global Microbiome Conservancy (https://microbiomeconservancy.org/), Kiel University, Germany.
⁶ Institute of Clinical and Molecular Biology, Kiel University, Germany.
⁷ Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, MA.
⁸ Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
⁹ Singapore-MIT Alliance for Research and Technology, Singapore.

PMID: 38895356
PMCID: PMC11185695
DOI: 10.1101/2024.06.03.597175

Update in

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, Berdy B, Bahunde JR, Girard C, Lehtimäki J, Mabulla AZP, Mwikarago IE, Nartey YA, Nguyen LTT, Onyekwere CA, Roberts LR, Shapiro BJ, Vatanen T, Comstock LE, Alm EJ, Dedon PC. Yuan Y, et al. Nat Commun. 2026 Jan 22;17(1):1717. doi: 10.1038/s41467-026-68412-5. Nat Commun. 2026. PMID: 41571653 Free PMC article.

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 uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX) brx genes, which were 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 6 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.

Keywords: comparative genomics; epigenetics; mass spectrometry; metagenomics; microbiome; next-generation sequencing; phosphorothioate.

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Figures

**Figure 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.

**Figure 2.. Gene neighborhoods analyses based on sequence similarity networks (SSNs) of DndC and SspD proteins essential for PT synthesis.**
SSN analysis was performed for the 3120 closest homologues of DndC (A) and 2132 homologues of SspD (B) in the UniProt database. Each node (black circle) in the network represents one DndC or SspD proteins. An edge (lines connecting nodes) is drawn between two nodes with a BLAST E-value cutoff of ≥10⁻¹⁰⁰ (alignment score of 100) in the DndC SSN or 10⁻⁶⁰ (alignment score of 60) in the SspD SSN. The node outlines are colored according to the presence of other *dnd* or *ssp* genes, with red outlines denoting colocation of *dndC* with *dndD* and similarly for *sspD* with *sspBC*. Some nodes are colored according to the type of genome neighborhood structure, which are represented below for species discussed in the text. For better visualization, single nodes and clusters with a few nodes were hidden. Abbreviations: NTPase, Nucleoside triphosphatase; MTase, methyltransferase; Res, restriction enzyme; HEPN, higher eukaryotes and prokaryotes nucleotide-binding.

**Figure 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. *ΔbrxC, ΔbrxC* pbrxC_Bf, *ΔbrxC* psspCBc, vector, and *brxCBs* are all significantly different from wilde-type (WT) by Student’s t-test, p <0.05.

**Figure 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 less than 5 genomes were hidden. Source data are provided as Table S5.

**Figure 5.. PT dinucleotides and PT consensus sequences in human gut microbiome isolates.**
The numbers of isolates were analyzed are listed on 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 on-going study. **, G_PSA and G_PST were detected using LC-MS QQQ but the exact mass was not verified by Orbitrap.

**Figure 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 1kb 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**) are 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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References

1. Sanchez-Romero M.A. & Casadesus J. The bacterial epigenome. Nat Rev Microbiol 18, 7–20 (2020). - PubMed
1. Thiaville J.J. et al. Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci U S A 113, E1452–1459 (2016). - PMC - PubMed
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This is a preprint.

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

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