Comparative Metabolomics Reveals the Production of Sulfated Metabolites by Human Gut Bacteria

The sulfated metabolome─the collection of sulfate-containing metabolites─is an emerging source of structurally unique bioactive compounds that influence metabolism, immune responses, and neurological function. Recent studies have shown that, in addition to host enzymes, gut bacteria also encode sulfotransferase enzymes (SULTs) that generate sulfated metabolites. However, the substrate scope of characterized gut bacterial SULTs remains narrow, and comprehensive discovery is limited by a lack of methods to detect and assign sulfated metabolites in complex samples. Here, we develop a comparative metabolomics workflow that leverages the universal SULT cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to incorporate heavy (34S) or light (32S) sulfur into sulfated metabolites, enabling discovery of microbiome-dependent sulfated compounds. By applying this approach in both "bottom-up" bacterial culture and "top-down" in vivo studies, we find that gut bacteria sulfonate hydroxy fatty acids. We identify a gut commensal microbe, Eubacterium ramulus, that performs this transformation, as well as an enzyme in this bacterium that performs this sulfonation, ErSULT. Metagenomic analyses reveal that ErSULT is prevalent across diverse human gut microbiomes. Together, this workflow and its application demonstrate that sulfated metabolite production by gut bacteria is more widespread than previously appreciated and provide a platform for future studies investigating the biosynthesis and biological functions of microbiome-derived sulfated small molecules.