Revealing CO₂-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO₂-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO₂-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their ¹³C-bicarbonate (¹³C-HCO₃^-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates ¹³C-HCO₃^-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO₂ fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling.