Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for >80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.

Carbon fixation by chemoautotrophic microorganisms in the dark ocean has a major impact on global carbon cycling and ecological relationships in the ocean's interior, but the relevant taxa and energy sources remain enigmatic. We show evidence that nitrite-oxidizing bacteria affiliated with the Nitrospinae phylum are important in dark ocean chemoautotrophy. Single-cell genomics and community metagenomics revealed that Nitrospinae are the most abundant and globally distributed nitrite-oxidizing bacteria in the ocean. Metaproteomics and metatranscriptomics analyses suggest that nitrite oxidation is the main pathway of energy production in Nitrospinae. Microautoradiography, linked with catalyzed reporter deposition fluorescence in situ hybridization, indicated that Nitrospinae fix 15 to 45% of inorganic carbon in the mesopelagic western North Atlantic. Nitrite oxidation may have a greater impact on the carbon cycle than previously assumed.

SignificanceCircumstantial evidence indicates that especially deep-ocean heterotrophic microbes rely on particulate organic matter sinking through the oceanic water column and being solubilized to dissolved organic matter (DOM) prior to utilization rather than on direct uptake of the vast pool of DOM in the deep ocean. Comparative metaproteomics allowed us to elucidate the vertical distribution and abundance of microbially mediated transport processes and thus the uptake of solutes throughout the oceanic water column. Taken together, our data suggest that, while the phylogenetic composition of the microbial community is depth stratified, the composition and substrate specificities of transporters considered in this study are ubiquitous while their relative abundance changes with depth.