Single-cell Amplified Genomes Research Articles

Metabolic Labeling and Digital Microfluidic Single-Cell Sequencing for Single Bacterial Genotypic-Phenotypic Analysis.

Accurate assessment of phenotypic and genotypic characteristics of bacteria can facilitate comprehensive cataloguing of all the resistance factors for better understanding of antibiotic resistance. However, current methods primarily focus on individual phenotypic or genotypic profiles across different colonies. Here, a Digital microfluidic-based automated assay for whole-genome sequencing of single-antibiotic-resistant bacteria is reported, enabling Genotypic and Phenotypic Analysis of antibiotic-resistant strains (Digital-GPA). Digital-GPA can efficiently isolate and sequence antibiotic-resistant bacteria illuminated by fluorescent D-amino acid (FDAA)-labeling, producing high-quality single-cell amplified genomes (SAGs). This enables identifications of both minor and major mutations, pinpointing substrains with distinctive resistance mechanisms. Digital-GPA can directly process clinical samples to detect and sequence resistant pathogens without bacterial culture, subsequently provide genetic profiles of antibiotic susceptibility, promising to expedite the analysis of hard-to-culture or slow-growing bacteria. Overall, Digital-GPA opens a new avenue for antibiotic resistance analysis by providing accurate and comprehensive molecular profiles of antibiotic resistance at single-cell resolution.

Survival Strategies of High GC-Content Microorganisms in Oligotrophic Deep Groundwater

Deep groundwaters are among the most energy and nutrient-limited ecosystems on the planet. The limited resources are mainly due to the absence of photosynthesis-driven primary production (Kadnikov et al. 2020). These ecosystems do however host phylogenetically diverse and metabolically active microorganisms from all domains of life plus viruses (Holmfeldt et al. 2021, Mehrshad et al. 2021). In this study, we used a large metagenomic dataset generated over the last eight years from the Äspö Hard Rock Laboratory (Äspö HRL) in Sweden and drill holes in Olkiluoto Island, Finland. This dataset, termed the “Fennoscandian Shield Genomic Dataset” (FSGD), contains metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs). Previous studies on this dataset have shown that reciprocal symbiotic partnerships and efficiency of energy metabolism define the core microbiome of these deep groundwaters (Mehrshad et al. 2021). Studies on different marine and freshwater ecosystems show that oligotrophic environments host streamlined genomes with lower GC content. However, it is not known how the low carbon and energy availability in deep groundwaters affect the microbial community regarding their genome size and GC content. To address this, we used the FSDG to study the distribution of genome size and GC content among bacterial and archaeal genomes in Fennoscandian Shield deep groundwaters. We further disentangled the prevalent metabolic strategies in these genomes that is being used to support their carbon and nitrogen demands for replication and survival. A total of 1990 MAGs/SAGs with a completeness of ≥50% and <5% contamination were recovered from 43 metagenomic datasets. The taxonomy of the MAGs/SAGs was assigned using the GTDB-tk (Chaumeil et al. 2022) and the GC content and genome size of MAGs/SAGs were calculated. MAGs/SAGs were also functionally annotated to investigate the genome-encoded functional potential. To survey the preference for different metabolic pathways and metabolic cross-feeding, the C-fixation pathways were subdivided into 82 routes that lead to the production of intermediate compounds (e.g., formate, pyruvate, oxaloacetate, etc.). A similar analysis for nitrogen acquisition pathways and CO2-capturing enzymes was performed and the presence/absence of these metabolic modules was investigated in representative MAGs/SAGs from various deep groundwater types to explore the microbial community’s metabolic interaction. The GC content and estimated genome size (EGS) of the recovered MAGs/SAGs were linearly correlated, suggesting that higher genome-level GC content is associated with larger genome size. The most common taxa among high GC (≥50%) content MAGs/SAGs were affiliated with the phyla Proteobacteria, Desulfobacterota, Actinobacteriota, Chloroflexota, and Patescibacteria. Analysis of metabolic modules in the environmental context revealed that high GC content MAGs constituted the main primary producers in all investigated groundwaters. Among the seven investigated C-fixation pathways, routes leading to the production of intermediates of reductive tricarboxylic acid (rTCA) were significantly enriched in high GC-content genomes. Both carboxylation/reduction first CO2-capturing enzymes were significantly enriched in MAGs with high GC contents. Of the various carbon fixation strategies, rGly and rTCA are the most energy-efficient and consume one and three ATP molecules, respectively (Bar-Even et al. 2011), matching the high prevalence of rTCA in oligotrophic deep groundwater. High GC content genomes featured metabolic pathways to generate key precursors for synthesizing purine/pyrimidine and amino acids. As GC base-pairs require one more nitrogen than AT base-pairs, prokaryotes with a higher GC content need proportionally more nitrogen for genome replication. In agreement with this, high GC content MAGs/SAGs from the FSGD feature additional nitrogen uptake systems including ammonium permease and N-fixation genes. High GC content MAGs/SAGs appear to be metabolically versatile and capable of acquiring nutrients via different carbon and nitrogen fixation pathways along with various C-scavenging strategies for substrate uptake to acquire energy and survive in oligotrophic conditions. Although their larger genome size and higher GC content entail higher expenses for replication and maintenance, their presence within the community of deep groundwater is supported by the “Black Queen Theory”. According to this theory, a cell containing an expensive metabolic pathway is maintained within the community as it provides a service that other members cannot.

Integrative Genomics Sheds Light on Global Deep Terrestrial Biosphere Communities

Active microbial lineages inhabit the deep terrestrial subsurface (Fry et al. 1997, Lopez-Fernandez et al. 2018) with estimates suggesting deep subsurface ecosystems to host ca. 90% of the total bacterial and archaeal biomass on Earth and about 10-20% of all terrestrial biomass (McMahon and Parnell 2014, Bar-On et al. 2018, Magnabosco et al. 2018, Drake and Reiners 2021). This highlights deep subsurface ecosystems as a vast reservoir of genetic innovation that remains poorly characterized. The biological diversity of deep subsurface ecosystems seem to be influenced by parameters such as temperature, pressure, water residence times, and geochemistry (Drake and Reiners 2021). The deep subsurface environment remains underrepresented in metagenomic databases, and yet the publicly available datasets are not synoptically analyzed in an integrated way. In this project, we collected a large set of publicly available and published metagenomes, published metagenome assembled genomes (MAGs) as well as single cell amplified genomes (SAGs) originating from global deep oligotrophic terrestrial subsurface samples (>70 m). Reconstructed MAGs/SAGs from this survey were integrated in to our own existing Fennoscandian Shield Genomic Database (FSGD) (Holmfeldt et al. 2021, Mehrshad et al. 2021). This expanded global database of the deep oligotrophic terrestrial subsurface contains >4000 MAGs/SAGs. In this dataset, representatives of phyla Firmicutes, Desulfobacterota, Patescibacteria, and Chlroflexota were the most prevalent taxa. The presence of Candidatus Desulforudis audaxviator across different continents and samples highlighted its adaptation to different deep biosphere environments. In addition, novel bacterial lineages at the class level were identified in several locations, pointing to the importance and necessity of further studies to characterize deep biosphere microbial diversity. This extensive study of the global deep oligotrophic terrestrial subsurface will reveal whether deep terrestrial populations are ubiquitous and how their distribution patterns are affected by different physicochemical variables such as water types and geology. By Identifying key populations present across multiple global groundwaters, our study will advance our understanding of the global diversity hosted in deep oligotrophic terrestrial subsurface ecosystems.

Disentangling hindgut metabolism in the American cockroach through single-cell genomics and metatranscriptomics.

Omnivorous cockroaches host a complex hindgut microbiota comprised of insect-specific lineages related to those found in mammalian omnivores. Many of these organisms have few cultured representatives, thereby limiting our ability to infer the functional capabilities of these microbes. Here we present a unique reference set of 96 high-quality single cell-amplified genomes (SAGs) from bacterial and archaeal cockroach gut symbionts. We additionally generated cockroach hindgut metagenomic and metatranscriptomic sequence libraries and mapped them to our SAGs. By combining these datasets, we are able to perform an in-depth phylogenetic and functional analysis to evaluate the abundance and activities of the taxa in vivo. Recovered lineages include key genera within Bacteroidota, including polysaccharide-degrading taxa from the genera Bacteroides, Dysgonomonas, and Parabacteroides, as well as a group of unclassified insect-associated Bacteroidales. We also recovered a phylogenetically diverse set of Firmicutes exhibiting a wide range of metabolic capabilities, including-but not limited to-polysaccharide and polypeptide degradation. Other functional groups exhibiting high relative activity in the metatranscriptomic dataset include multiple putative sulfate reducers belonging to families in the Desulfobacterota phylum and two groups of methanogenic archaea. Together, this work provides a valuable reference set with new insights into the functional specializations of insect gut symbionts and frames future studies of cockroach hindgut metabolism.

A compendium of bacterial and archaeal single-cell amplified genomes from oxygen deficient marine waters

Oxygen-deficient marine waters referred to as oxygen minimum zones (OMZs) or anoxic marine zones (AMZs) are common oceanographic features. They host both cosmopolitan and endemic microorganisms adapted to low oxygen conditions. Microbial metabolic interactions within OMZs and AMZs drive coupled biogeochemical cycles resulting in nitrogen loss and climate active trace gas production and consumption. Global warming is causing oxygen-deficient waters to expand and intensify. Therefore, studies focused on microbial communities inhabiting oxygen-deficient regions are necessary to both monitor and model the impacts of climate change on marine ecosystem functions and services. Here we present a compendium of 5,129 single-cell amplified genomes (SAGs) from marine environments encompassing representative OMZ and AMZ geochemical profiles. Of these, 3,570 SAGs have been sequenced to different levels of completion, providing a strain-resolved perspective on the genomic content and potential metabolic interactions within OMZ and AMZ microbiomes. Hierarchical clustering confirmed that samples from similar oxygen concentrations and geographic regions also had analogous taxonomic compositions, providing a coherent framework for comparative community analysis.

Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral

BackgroundEndozoicomonas bacteria symbiosis with various marine organisms is hypothesized as a potential indicator of health in corals. Although many amplicon analyses using 16S rRNA gene have suggested the diversity of Endozoicomonas species, genome analysis has been limited due to contamination of host-derived sequences and difficulties in culture and metagenomic analysis. Therefore, the evolutionary and functional potential of individual Endozoicomonas species symbiotic with the same coral species remains unresolved.ResultsIn this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genomes (SAGs) for uncultured coral-associated Endozoicomonas spp. We obtained seven novel Endozoicomonas genomes and quantitative bacterial composition from Acropora tenuis corals at four sites in Japan. Our quantitative 16S rRNA gene and comparative genomic analysis revealed that these Endozoicomonas spp. belong to different lineages (Clade A and Clade B), with widely varying abundance among individual corals. Furthermore, each Endozoicomonas species possessed various eukaryotic-like genes in clade-specific genes. It was suggested that these eukaryotic-like genes might have a potential ability of different functions in each clade, such as infection of the host coral or suppression of host immune pathways. These Endozoicomonas species may have adopted different host adaptation strategies despite living symbiotically on the same coral.ConclusionsThis study suggests that coral-associated Endozoicomonas spp. on the same species of coral have different evolutional strategies and functional potentials in each species and emphasizes the need to analyze the genome of each uncultured strain in future coral-Endozoicomonas relationships studies.BqfFwXzdNKfPeBMVktWjTuVideo

It is time for a new type of type to facilitate naming the microbial world

Since January 1, 2001, the only acceptable nomenclatural type for species under the International Code of Nomenclature of Prokaryotes (ICNP) has been pure cultures. Here, we argue that this requirement is discordant with the more inclusive nature of nomenclatural types accepted under other codes of nomenclature and posit that the unique rigidity of the ICNP has failed to serve the broad research community and has stifled progress. This case is based on the axiom that many archaea and bacteria are interdependent in nature and therefore difficult, if not impossible, to grow, preserve, and distribute as pure cultures. As such, a large proportion of Earth's biodiversity cannot be named under the current system, which limits our ability to communicate about microbial diversity within and beyond the microbiology research community. Genome sequence data are now encouraged for valid publication of new taxa in microbial systematics journals, and metagenome-assembled genomes and single cell-amplified genomes are being generated rapidly from every biome on Earth. Thus, genome sequences are available for both cultivated and uncultivated microorganisms and can readily serve as a new category of nomenclatural type, allowing for a unified nomenclature for all archaea and bacteria, whether or not they are available as pure cultures. Ideally this would be under a single code of nomenclature but, as we review here, the newly established SeqCode will operate in parallel with the ICNP as a first step toward this goal.

Comparative genome analysis of the genus Hydrotalea and proposal of the novel species Hydrotalea lipotrueae sp. nov., isolated from a groundwater aquifer in the south of Mallorca Island, Spain

From a collection of > 140 strains isolated from groundwater with thermal anomalies for the purpose of obtaining good candidates with applications in the cosmetic industry, two strains were selected because of their taxonomic novelty. Among the isolates, strains TMF_100T and TFM_099 stood out for their potential biotechnological relevance, and a comparative analysis of 16S rRNA gene sequences indicated that these strains represented a new species of the genus Hydrotalea. In addition, from the public genomic databases, metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) could be retrieved that affiliated with this genus. These MAGs and SAGs had been obtained from different environmental samples, such as acid mine drainage or marine sediments. In addition to the description of the new species, the ecological relevance of the members of this genus was demonstrated by means of denitrification, CRISPR-Cas system diversity and heavy metal resistance, as well as their wide geographical distribution and environmental versatility. Supported by the taxonomic study, together with physiological and morphological differences and ecological features, we concluded that strain TMF_100T represented a novel species within the genus Hydrotalea, for which we propose the name Hydrotalea lipotrueae sp. nov.

Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics

BackgroundObtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once.ResultsOur sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis).ConclusionsSMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker.3Xqp2i4EXkShdVYdkpPFdnVideo abstract

Genomic Variation Influences Methanothermococcus Fitness in Marine Hydrothermal Systems.

Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans

BackgroundThe supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered.ResultsWe sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome.ConclusionsOur data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

Description of Candidatus Mesopelagibacter carboxydoxydans and Candidatus Anoxipelagibacter denitrificans: Nitrate-reducing SAR11 genera that dominate mesopelagic and anoxic marine zones

The diverse and ubiquitous members of the SAR11 lineage (Alphaproteobacteria) represent up to 30–40% of the surface and mesopelagic oceanic microbial communities. However, the molecular and ecological mechanisms that differentiate closely related, yet distinct, SAR11 members that often co-occur under similar environmental conditions remain speculative. Recently, two mesopelagic and oxygen minimum zone (OMZ)-associated subclades of SAR11 (Ic and IIa.A) were described using single-cell amplified genomes (SAGs) linked to nitrate reduction in OMZs. In this current study, the collection of genomes belonging to these two subclades was expanded with thirteen new metagenome-assembled genomes (MAGs), thus providing a more detailed phylogenetic and functional characterization of these subclades. Gene content-based predictions of metabolic functions revealed similarities in central carbon metabolism between subclades Ic and IIa.A and surface SAR11 clades, with small variations in central pathways. These variations included more versatile sulfur assimilation pathways, as well as a previously predicted capacity for nitrate reduction that conferred unique versatility on mesopelagic-adapted clades compared to their surface counterparts. Finally, consistent with previously reported abundances of carbon monoxide (CO) in surface and mesopelagic waters, subclades Ia (surface) and Ic (mesopelagic) have the genetic potential to oxidize carbon monoxide (CO), presumably taking advantage of this abundant compound as an electron donor. Based on genomic analyses, environmental distribution and metabolic reconstruction, we propose two new SAR11 genera, Ca. Mesopelagibacter carboxydoxydans (subclade Ic) and Ca. Anoxipelagibacter denitrificans (subclade IIa.A), which represent members of the mesopelagic and OMZ-adapted SAR11 clades.

Genomic Characteristics Distinguish Geographically Distributed Dehalococcoidia.

Dehalococcoidia (Dia) class microorganisms are frequently found in various pristine and contaminated environments. Metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) studies have substantially improved the understanding of Dia microbial ecology and evolution; however, an updated thorough investigation on the genomic and evolutionary characteristics of Dia microorganisms distributed in geographically distinct environments has not been implemented. In this study, we analyzed available genomic data to unravel Dia evolutionary and metabolic traits. Based on the phylogeny of 16S rRNA genes retrieved from sixty-seven genomes, Dia microorganisms can be categorized into three groups, the terrestrial cluster that contains all Dehalococcoides and Dehalogenimonas strains, the marine cluster I, and the marine cluster II. These results reveal that a higher ratio of horizontally transferred genetic materials was found in the Dia marine clusters compared to that of the Dia terrestrial cluster. Pangenome analysis further suggests that Dia microorganisms have evolved cluster-specific enzymes (e.g., dehalogenase in terrestrial Dia, sulfite reductase in marine Dia) and biosynthesis capabilities (e.g., siroheme biosynthesis in marine Dia). Marine Dia microorganisms are likely adapted to versatile metabolisms for energy conservation besides organohalide respiration. The genomic differences between marine and terrestrial Dia may suggest distinct functions and roles in element cycling (e.g., carbon, sulfur, chlorine), which require interdisciplinary approaches to unravel the physiology and evolution of Dia in various environments.

Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.

Single cell ecogenomics reveals mating types of individual cells and ssDNA viral infections in the smallest photosynthetic eukaryotes.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13-22 Mb genomes from three genera, Ostreococcus, Bathycoccus and Micromonas, are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue 'Single cell ecology'.

MiComplete: weighted quality evaluation of assembled microbial genomes.

Metagenomics and single-cell genomics have revolutionized the study of microorganisms, increasing our knowledge of microbial genomic diversity by orders of magnitude. A major issue pertaining to metagenome-assembled genomes (MAGs) and single-cell amplified genomes (SAGs) is to estimate their completeness and redundancy. Most approaches rely on counting conserved gene markers. In miComplete, we introduce a weighting strategy, where we normalize the presence/absence of markers by their median distance to the next marker in a set of complete reference genomes. This approach alleviates biases introduced by the presence/absence of shorter DNA pieces containing many markers, e.g. ribosomal protein operons. miComplete is written in Python 3 and released under GPLv3. Source code and documentation are available at https://bitbucket.org/evolegiolab/micomplete. Supplementary data are available at Bioinformatics online.

Uncultured Microbial Phyla Suggest Mechanisms for Multi-Thousand-Year Subsistence in Baltic Sea Sediments.

Energy-starved microbes in deep marine sediments subsist at near-zero growth for thousands of years, yet the mechanisms for their subsistence are unknown because no model strains have been cultivated from most of these groups. We investigated Baltic Sea sediments with single-cell genomics, metabolomics, metatranscriptomics, and enzyme assays to identify possible subsistence mechanisms employed by uncultured Atribacteria, Aminicenantes, Actinobacteria group OPB41, Aerophobetes, Chloroflexi, Deltaproteobacteria, Desulfatiglans, Bathyarchaeota, and Euryarchaeota marine group II lineages. Some functions appeared to be shared by multiple lineages, such as trehalose production and NAD+-consuming deacetylation, both of which have been shown to increase cellular life spans in other organisms by stabilizing proteins and nucleic acids, respectively. Other possible subsistence mechanisms differed between lineages, possibly providing them different physiological niches. Enzyme assays and transcripts suggested that Atribacteria and Actinobacteria group OPB41 catabolized sugars, whereas Aminicenantes and Atribacteria catabolized peptides. Metabolite and transcript data suggested that Atribacteria utilized allantoin, possibly as an energetic substrate or chemical protectant, and also possessed energy-efficient sodium pumps. Atribacteria single-cell amplified genomes (SAGs) recruited transcripts for full pathways for the production of all 20 canonical amino acids, and the gene for amino acid exporter YddG was one of their most highly transcribed genes, suggesting that they may benefit from metabolic interdependence with other cells. Subsistence of uncultured phyla in deep subsurface sediments may occur through shared strategies of using chemical protectants for biomolecular stabilization, but also by differentiating into physiological niches and metabolic interdependencies.IMPORTANCE Much of life on Earth exists in a very slow-growing state, with microbes from deeply buried marine sediments representing an extreme example. These environments are like natural laboratories that have run multi-thousand-year experiments that are impossible to perform in a laboratory. We borrowed some techniques that are commonly used in laboratory experiments and applied them to these natural samples to make hypotheses about how these microbes subsist for so long at low activity. We found that some methods for stabilizing proteins and nucleic acids might be used by many members of the community. We also found evidence for niche differentiation strategies, and possibly cross-feeding, suggesting that even though they are barely growing, complex ecological interactions continue to occur over ultralong timescales.

Metabolic potential and in situ transcriptomic profiles of previously uncharacterized key microbial groups involved in coupled carbon, nitrogen and sulfur cycling in anoxic marine zones.

Anoxic marine zones (AMZs) impact biogeochemical cycles at the global scale, particularly the nitrogen cycle. Key microbial players from AMZs have been identified, but the majority remains unrecognized or uncharacterized. Thirty-one single-cell amplified genomes (SAGs) from the eastern tropical North and South Pacific AMZs were sequenced to gain insight into the distribution, metabolic potential and contribution to the community transcriptional profile of these uncharacterized bacterial and archaeal groups. Detailed analyses focused on SAG-bins assigned to three of these groups that presented 79%-100% estimated genome completeness: the putative sulphur-oxidizing Gamaproteobacteria EOSA II clade, a Marinimicrobia member of the recently recognized PN262000N21 clade found to be abundant in AMZ anoxic cores, and a representative of the Marine Benthic Group A Thaumarchaeota. Community-based analyses revealed that these three groups are significantly more abundant and transcriptionally more active in the AMZ microbial communities than previously described phylogenetically related microbial groups. Collectively, these groups have the potential to link biogeochemically relevant processes by coupling the carbon, nitrogen and sulfur cycles. Together, these results increase our understanding of key microbial components inhabiting AMZs and other oxygen-deficient marine environments, enhancing our capacity to predict the impact of the expansion of these ecosystems due to climate change.

Obtaining high-quality draft genomes from uncultured microbes by cleaning and co-assembly of single-cell amplified genomes

Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

Evaluation of single-cell genomics to address evolutionary questions using three SAGs of the choanoflagellate Monosiga brevicollis

Single-cell genomics (SCG) appeared as a powerful technique to get genomic information from uncultured organisms. However, SCG techniques suffer from biases at the whole genome amplification step that can lead to extremely variable numbers of genome recovery (5–100%). Thus, it is unclear how useful can SCG be to address evolutionary questions on uncultured microbial eukaryotes. To provide some insights into this, we here analysed 3 single-cell amplified genomes (SAGs) of the choanoflagellate Monosiga brevicollis, whose genome is known. Our results show that each SAG has a different, independent bias, yielding different levels of genome recovery for each cell (6–36%). Genes often appear fragmented and are split into more genes during annotation. Thus, analyses of gene gain and losses, gene architectures, synteny and other genomic features can not be addressed with a single SAG. However, the recovery of phylogenetically-informative protein domains can be up to 55%. This means SAG data can be used to perform accurate phylogenomic analyses. Finally, we also confirm that the co-assembly of several SAGs improves the general genomic recovery. Overall, our data show that, besides important current limitations, SAGs can still provide interesting and novel insights from poorly-known, uncultured organisms.