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.