Byrne Shane R, DeMott Michael S, Yuan Yifeng, Ghanegolmohammadi Farzan, Kaiser Stefanie, Fox James G, Alm Eric J, Dedon Peter C
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
Microbiome. 2025 Mar 24;13(1):81. doi: 10.1186/s40168-025-02071-4.
Epigenetic regulation of gene expression and host defense is well established in microbial communities, with dozens of DNA modifications comprising the epigenomes of prokaryotes and bacteriophage. Phosphorothioation (PT) of DNA, in which a chemically reactive sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is catalyzed by dnd and ssp gene families widespread in bacteria and archaea. However, little is known about the role of PTs or other microbial epigenetic modifications in the human microbiome. Here we optimized and applied fecal DNA extraction, mass spectrometric, and metagenomics technologies to characterize the landscape and temporal dynamics of gut microbes possessing PT modifications.
Exploiting the nuclease-resistance of PTs, mass spectrometric analysis of limit digests of PT-containing DNA reveals PT dinucleotides as part of genomic consensus sequences, with 16 possible dinucleotide combinations. Analysis of mouse fecal DNA revealed a highly uniform spectrum of 11 PT dinucleotides in all littermates, with PTs estimated to occur in 5-10% of gut microbes. Though at similar levels, PT dinucleotides in fecal DNA from 11 healthy humans possessed signature combinations and levels of individual PTs. Comparison with a widely distributed microbial epigenetic mark, mdA, suggested temporal dynamics consistent with expectations for gut microbial communities based on Taylor's Power Law. Application of PT-seq for site-specific metagenomic analysis of PT-containing bacteria in one fecal donor revealed the larger consensus sequences for the PT dinucleotides in Bacteroidota, Bacillota (formerly Firmicutes), Actinomycetota (formerly Actinobacteria), and Pseudomonadota (formerly Proteobacteria), which differed from unbiased metagenomics and suggested that the abundance of PT-containing bacteria did not simply mirror the spectrum of gut bacteria. PT-seq further revealed low abundance PT sites not detected as dinucleotides by mass spectrometry, attesting to the complementarity of the technologies. Video Abstract CONCLUSIONS: The results of our studies provide a benchmark for understanding the behavior of an abundant and chemically reactive epigenetic mark in the human gut microbiome, with implications for inflammatory conditions of the gut.
基因表达和宿主防御的表观遗传调控在微生物群落中已得到充分证实，数十种DNA修饰构成了原核生物和噬菌体的表观基因组。DNA的硫代磷酸化（PT），即一个化学反应性硫原子取代糖磷酸骨架中的非桥连氧，由广泛存在于细菌和古菌中的dnd和ssp基因家族催化。然而，关于PT或其他微生物表观遗传修饰在人类微生物组中的作用知之甚少。在这里，我们优化并应用了粪便DNA提取、质谱和宏基因组学技术，以表征具有PT修饰的肠道微生物的格局和时间动态。
利用PT对核酸酶的抗性，对含PT的DNA进行有限消化后的质谱分析揭示了PT二核苷酸作为基因组共有序列的一部分，有16种可能的二核苷酸组合。对小鼠粪便DNA的分析显示，所有同窝小鼠中11种PT二核苷酸的谱图高度一致，估计PT存在于5-10%的肠道微生物中。虽然水平相似，但来自11名健康人类的粪便DNA中的PT二核苷酸具有独特的组合和个体PT水平。与广泛分布的微生物表观遗传标记mdA的比较表明，时间动态符合基于泰勒幂律对肠道微生物群落的预期。应用PT-seq对一名粪便供体中含PT细菌进行位点特异性宏基因组分析，揭示了拟杆菌门、芽孢杆菌门（原厚壁菌门）、放线菌门（原放线菌）和假单胞菌门（原变形菌门）中PT二核苷酸的更大共有序列，这与无偏差宏基因组学不同，表明含PT细菌的丰度并非简单反映肠道细菌的谱图。PT-seq进一步揭示了质谱未检测为二核苷酸的低丰度PT位点，证明了这些技术的互补性。视频摘要
我们的研究结果为理解人类肠道微生物组中一种丰富且具有化学反应性的表观遗传标记的行为提供了一个基准，对肠道炎症状况具有启示意义。
