DSMG-Chip: A High-Throughput Degenerate qPCR Chip for Profiling Microbial DMSP and Related Organic Sulfur Metabolic Genes in Diverse Environments

The organosulfur cycle involves active microbial transformations of dimethylsulfoniopropionate (DMSP) to yield the climate active gas dimethyl sulfide (DMS) and other compounds. The lack of rapid and accurate methods to quantify the DMSP metabolic potential hinders a deeper understanding of this cycling. We developed a high-throughput qPCR (HT-qPCR) chip, DSMG-chip, to quantify the absolute abundance of DMSP and related organic sulfur metabolic genes. DSMG-chip contains 42 degenerate primer sets targeting 27 organosulfur metabolic genes, with the 16S rRNA gene as a reference, allowing for the detection of 41 environmental samples simultaneously. In silico analysis indicated that the DSMG-chip possesses broad taxonomic coverage (1.4-91.3%, spanning 12 phyla and 275 genera) and high specificity (44.4-100%, mean: 85.34%). Validation experiments using conventional PCR, qPCR, and HT-qPCR confirmed the primers' strict specificity, robust amplification efficiency (0.677 to 0.997, mean: 0.771), and excellent accuracy, correlating well with conventional qPCR (Pearson's r = 0.914). Finally, application of the DSMG-chip accurately reflected variations in DMSP metabolism across diverse seawater and sediment samples, highlighting the active microbial DMSP cycling in Pacific Ocean (section P) seawater and the contribution of the methanethiol (MeSH) methylation pathway (via mddA) in sediments. Our findings provide a powerful, novel tool for studying the organosulfur cycle.