Phylogeny-metabolism dual-directed single-cell genomics for dissecting and mining ecosystem function

Abstract

AbstractAlthough microbiome-wide association studies (MWAS) have uncovered many marker organisms for an ecosystem trait, mechanisms of most microbiota-mediated processes remain elusive, due to challenges in validating the markers’in situmetabolic activities and tracing such activities to individual genomes. Here we introduced a phylogeny-metabolism dual-directed single-cell genomics approach called Fluorescence-In-Situ-Hybridization-guided Single-Cell Raman-activated Sorting and Sequencing (FISH-scRACS-Seq). It directly localizes individual cells from target taxon via a FISH probe for marker organism, profiles theirin situmetabolic functions via single-cell Raman spectra, sorts cells of target taxonomy and target metabolism, and produces indexed, high-coverage and precisely-one-cell genomes. From cyclohexane-contaminated seawater, cells representing the MWAS-derived marker taxon of γ-Proteobacteria and that are actively degrading cyclohexanein situwere directly identified via FISH and Raman respectively, then sorted and sequenced for one-cell full genomes. In such aPseudoalteromonas fuligineacell, we discovered a three-component cytochrome P450 system that can convert cyclohexane to cyclohexanolin vitro, representing a previously unknown group of cyclohexane-degrading enzymes and organisms. By culture-independently unveiling enzymes, pathways, genomes and theirin situfunctions specifically for those single-cells with ecological relevance, FISH-scRACS-Seq is a rational and generally applicable approach for dissecting and mining microbiota functions.TeaserFISH-scRACS-Seq is a new strategy to dissect microbiota functional mechanism at single-cell resolution.