One-Cell Metabolic Phenotyping and Sequencing of Soil Microbiome by Raman-Activated Gravity-Driven Encapsulation (RAGE)

Abstract

Soil harbors arguably the most metabolically and genetically heterogeneous microbiomes on Earth, yet establishing the link between metabolic functions and genome at the precisely one-cell level has been difficult. Here, for mock microbial communities and then for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) platform, which identifies, sorts, and sequences precisely one bacterial cell via its anabolic (incorporating D from heavy water) and physiological (carotenoid-containing) functions. We showed that (i) metabolically active cells from numerically rare soil taxa, such as <i>Corynebacterium</i> spp., <i>Clostridium</i> spp., <i>Moraxella</i> spp., <i>Pantoea</i> spp., and Pseudomonas spp., can be readily identified and sorted based on D₂O uptake, and their one-cell genome coverage can reach ∼93% to allow high-quality genome-wide metabolic reconstruction; (ii) similarly, carotenoid-containing cells such as <i>Pantoea</i> spp., <i>Legionella</i> spp., <i>Massilia</i> spp., Pseudomonas spp., and <i>Pedobacter</i> spp. were identified and one-cell genomes were generated for tracing the carotenoid-synthetic pathways; and (iii) carotenoid-producing cells can be either metabolically active or inert, suggesting culture-based approaches can miss many such cells. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at exactly one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems. <b>IMPORTANCE</b> Soil is home to an enormous and complex microbiome that features arguably the highest genomic diversity and metabolic heterogeneity of cells on Earth. Their <i>in situ</i> metabolic activities drive many natural processes of pivotal ecological significance or underlie industrial production of numerous valuable bioactivities. However, pinpointing "who is doing what" in a soil microbiome, which consists of mainly yet-to-be-cultured species, has remained a major challenge. Here, for soil microbiota, we established a Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq) method, which identifies, sorts, and sequences at the resolution of precisely one microbial cell via its catabolic and anabolic functions. As a Raman-activated cell sorter (RACS) family member that can establish a metabolism-genome link at one-cell resolution from soil, RAGE-Seq can help to precisely pinpoint "who is doing what" in complex ecosystems.