Charting the Complexity of the Marine Microbiome through Single-Cell Genomics.

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.

Decoupling of respiration rates and abundance in marine prokaryoplankton.

The ocean-atmosphere exchange of CO2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton)1-3, their respiration usually is measured in bulk and treated as a 'black box' in global biogeochemical models4; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy3,5-7 probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO2 for its energy demands and growth may be lower than commonly assumed and variable among lineages.

Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem.

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~103 cells mL-1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator.

Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Radiometric approach for the detection of picophytoplankton assemblages across oceanic fronts.

Cell abundances of Prochlorococcus, Synechococcus, and autotrophic picoeukaryotes were estimated in surface waters using principal component analysis (PCA) of hyperspectral and multispectral remote-sensing reflectance data. This involved the development of models that employed multilinear correlations between cell abundances across the Atlantic Ocean and a combination of PCA scores and sea surface temperatures. The models retrieve high Prochlorococcus abundances in the Equatorial Convergence Zone and show their numerical dominance in oceanic gyres, with decreases in Prochlorococcus abundances towards temperate waters where Synechococcus flourishes, and an emergence of picoeukaryotes in temperate waters. Fine-scale in-situ sampling across ocean fronts provided a large dynamic range of measurements for the training dataset, which resulted in the successful detection of fine-scale Synechococcus patches. Satellite implementation of the models showed good performance (R2 > 0.50) when validated against in-situ data from six Atlantic Meridional Transect cruises. The improved relative performance of the hyperspectral models highlights the importance of future high spectral resolution satellite instruments, such as the NASA PACE mission's Ocean Color Instrument, to extend our spatiotemporal knowledge about ecologically relevant phytoplankton assemblages.

Genome to phenome tools: In vivo and in vitro transfection of Crassostrea virginica hemocytes.

The sequencing of the Crassostrea virginica genome has brought back the interest for gene delivery and editing methodologies. Here, we report the expression in oyster hemocytes of two heterologous expression vectors under the CMV promoter delivered with dendrimers. Expression was monitored using confocal microscopy, flow cytometry, and immunofluorescence assay. C. virginica hemocytes were able to express the green fluorescence protein and Crassostrea gigas vascular endothelial growth factor under CMV viral promoter both in vivo and in vitro. These results provide the bases for interrogating the genome and adapting genome editing methodologies.

Accumulation and enhanced cycling of polyphosphate by Sargasso Sea plankton in response to low phosphorus.

Phytoplankton alter their biochemical composition according to nutrient availability, such that their bulk elemental composition varies across oceanic provinces. However, the links between plankton biochemical composition and variation in biogeochemical cycling of nutrients remain largely unknown. In a survey of phytoplankton phosphorus stress in the western North Atlantic, we found that phytoplankton in the phosphorus-depleted subtropical Sargasso Sea were enriched in the biochemical polyphosphate (polyP) compared with nutrient-rich temperate waters, contradicting the canonical oceanographic view of polyP as a luxury phosphorus storage molecule. The enrichment in polyP coincided with enhanced alkaline phosphatase activity and substitution of sulfolipids for phospholipids, which are both indicators of phosphorus stress. Further, polyP appeared to be liberated preferentially over bulk phosphorus from sinking particles in the Sargasso Sea, thereby retaining phosphorus in shallow waters. Thus, polyP cycling may form a feedback loop that attenuates the export of phosphorus when it becomes scarce, contributes bioavailable P for primary production, and supports the export of carbon and nitrogen via sinking particles.

Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean.

Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.

Oceanic Crustal Fluid Single Cell Genomics Complements Metagenomic and Metatranscriptomic Surveys With Orders of Magnitude Less Sample Volume.

Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 103 cells ml-1) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to 'omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis.

Single Cell Genomics Reveals Viruses Consumed by Marine Protists.

The predominant model of the role of viruses in the marine trophic web is that of the "viral shunt," where viral infection funnels a substantial fraction of the microbial primary and secondary production back to the pool of dissolved organic matter. Here, we analyzed the composition of non-eukaryotic DNA associated with individual cells of small, planktonic protists in the Gulf of Maine (GoM) and the Mediterranean Sea. We found viral DNA associated with a substantial fraction cells from the GoM (51%) and the Mediterranean Sea (35%). While Mediterranean SAGs contained a larger proportion of cells containing bacterial sequences (49%), a smaller fraction of cells contained bacterial sequences in the GoM (19%). In GoM cells, nearly identical bacteriophage and ssDNA virus sequences where found across diverse lineages of protists, suggesting many of these viruses are non-infective. The fraction of cells containing viral DNA varied among protistan lineages and reached 100% in Picozoa and Choanozoa. These two groups also contained significantly higher numbers of viral sequences than other identified taxa. We consider mechanisms that may explain the presence of viral DNA in protistan cells and conclude that protistan predation on free viral particles contributed to the observed patterns. These findings confirm prior experiments with protistan isolates and indicate that the viral shunt is complemented by a viral link in the marine microbial food web. This link may constitute a sink of viral particles in the ocean and has implications for the flow of carbon through the microbial food web.

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

Temporal dynamics of eukaryotic microbial diversity at a coastal Pacific site.

High-throughput sequencing of ocean biomes has revealed vast eukaryotic microbial diversity, a significant proportion of which remains uncharacterized. Here we use a temporal approach to understanding eukaryotic diversity at the Scripps Pier, La Jolla, California, USA, via high-throughput amplicon sequencing of the 18S rRNA gene, the abundances of both Synechococcus and Synechococcus grazers, and traditional oceanographic parameters. We also exploit our ability to track operational taxonomic units (OTUs) temporally to evaluate the ability of 18S sequence-based OTU assignments to meaningfully reflect ecological dynamics. The eukaryotic community is highly dynamic in terms of both species richness and composition, although proportional representation of higher-order taxa remains fairly consistent over time. Synechococcus abundance fluctuates throughout the year. OTUs unique to dates of Synechococcus blooms and crashes or enriched in Synechococcus addition incubation experiments suggest that the prasinophyte Tetraselmis sp. and Gymnodinium-like dinoflagellates are likely Synechococcus grazers under certain conditions, and may play an important role in their population fluctuations.

Genomic exploration of individual giant ocean viruses.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

Author Correction: Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

The original version of this Article contained errors in the units of concentration of three reagents listed in the Methods. These errors have all been corrected in both the PDF and HTML versions of the Article.

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

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.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.

High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton.

Recent discoveries suggest that photoheterotrophs (rhodopsin-containing bacteria (RBs) and aerobic anoxygenic phototrophs (AAPs)) and chemoautotrophs may be significant for marine and freshwater ecosystem productivity. However, their abundance and taxonomic identities remain largely unknown. We used a combination of single-cell and metagenomic DNA sequencing to study the predominant photoheterotrophs and chemoautotrophs inhabiting the euphotic zone of temperate, physicochemically diverse freshwater lakes. Multi-locus sequencing of 712 single amplified genomes, generated by fluorescence-activated cell sorting and whole genome multiple displacement amplification, showed that most of the cosmopolitan freshwater clusters contain photoheterotrophs. These comprised at least 10-23% of bacterioplankton, and RBs were the dominant fraction. Our data demonstrate that Actinobacteria, including clusters acI, Luna and acSTL, are the predominant freshwater RBs. We significantly broaden the known taxonomic range of freshwater RBs, to include Alpha-, Beta-, Gamma- and Deltaproteobacteria, Verrucomicrobia and Sphingobacteria. By sequencing single cells, we found evidence for inter-phyla horizontal gene transfer and recombination of rhodopsin genes and identified specific taxonomic groups involved in these evolutionary processes. Our data suggest that members of the ubiquitous betaproteobacteria Polynucleobacter spp. are the dominant AAPs in temperate freshwater lakes. Furthermore, the RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) gene was found in several single cells of Betaproteobacteria, Bacteroidetes and Gammaproteobacteria, suggesting that chemoautotrophs may be more prevalent among aerobic bacterioplankton than previously thought. This study demonstrates the power of single-cell DNA sequencing addressing previously unresolved questions about the metabolic potential and evolutionary histories of uncultured microorganisms, which dominate most natural environments.

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing.

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator-prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.

Single cell genome analysis supports a link between phagotrophy and primary plastid endosymbiosis.

Two cases of primary plastid endosymbiosis are known. The first occurred ca. 1.6 billion years ago and putatively gave rise to the canonical plastid in algae and plants. The second is restricted to a genus of rhizarian amoebae that includes Paulinella chromatophora. Photosynthetic Paulinella species gained their plastid from an α-cyanobacterial source and are sister to plastid-lacking phagotrophs such as Paulinella ovalis that ingest cyanobacteria. To study the role of feeding behavior in plastid origin, we analyzed single-cell genome assemblies from six P. ovalis-like cells isolated from Chesapeake Bay, USA. Dozens of contigs in these cell assemblies were derived from prey DNA of α-cyanobacterial origin and associated cyanophages. We found two examples of horizontal gene transfer (HGT) in P. ovalis-like nuclear DNA from cyanobacterial sources. This work suggests the first evidence of a link between feeding behavior in wild-caught cells, HGT, and plastid primary endosymbiosis in the monophyletic Paulinella lineage.