[show abstract][hide abstract] ABSTRACT: The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet.
We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules.
We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host.
[show abstract][hide abstract] ABSTRACT: Previous studies based on analysis of amoA, 16S ribosomal RNA or accA gene sequences have established that marine Thaumarchaeota fall into two phylogenetically distinct groups corresponding to shallow- and deep-water clades, but it is not clear how water depth interacts with other environmental factors, including light, temperature and location, to affect this pattern of diversification. Earlier studies focused on single-gene distributions were not able to link phylogenetic structure to other aspects of functional adaptation. Here, we analyzed the genome content of 46 uncultivated single Thaumarchaeota cells sampled from epi- and mesopelagic waters of subtropical, temperate and polar oceans. Phylogenomic analysis showed that populations diverged by depth, as expected, and that mesopelagic populations from different locations were well mixed. Functional analysis showed that some traits, including putative DNA photolyase and catalase genes that may be related to adaptive mechanisms to reduce light-induced damage, were found exclusively in members of the epipelagic clade. Our analysis of partial genomes has thus confirmed the depth differentiation of Thaumarchaeota populations observed previously, consistent with the distribution of putative mechanisms to reduce light-induced damage in shallow- and deep-water populations.The ISME Journal advance online publication, 7 November 2013; doi:10.1038/ismej.2013.202.
[show abstract][hide abstract] ABSTRACT: Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared to freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities.
[show abstract][hide abstract] ABSTRACT: Abstract Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing "microbial dark matter" that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license.
Journal of computational biology: a journal of computational molecular cell biology 10/2013; 20(10):714-737. · 1.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated 'DEH-J10' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO2 or oxidation of organics completely to CO2. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.The ISME Journal advance online publication, 22 August 2013; doi:10.1038/ismej.2013.143.
[show abstract][hide abstract] ABSTRACT: Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal
stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.
[show abstract][hide abstract] ABSTRACT: Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 06/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Half of the microbial cells in the Earth's oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.
[show abstract][hide abstract] ABSTRACT: Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32-35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola's community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.
[show abstract][hide abstract] ABSTRACT: Single cell genomics (SCG) uncovers hereditary information at the most basic level of biological organization. It is emerging as a powerful complement to cultivation-based and microbial community-focused research approaches. SCG has been instrumental in identifying metabolic features, evolutionary histories and inter-organismal interactions of the uncultured microbial groups that dominate many environments and biogeochemical cycles. The SCG approach also holds great promise in microbial microevolution studies and industrial bioprospecting. Methods for SCG consist of a series of integrated processes, beginning with the collection and preservation of environmental samples, followed by physical separation, lysis and whole genome amplification of individual cells, and culminating in genomic sequencing and the inference of encoded biological features.
Current opinion in microbiology 09/2012; · 7.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.
[show abstract][hide abstract] ABSTRACT: Concentrations of pelagic fungal biomass, determined as the content of ergosterol in particulate matter, were measured in
49 Baltic rivers during summer 1999. The ergosterol concentration varied 12-fold, from 12.6 to 152.5ngl−1 (average of 56.4ngl−1) and correlated positively with concentrations of dissolved organic matter and inorganic nutrients as well as with spectral
DOM properties indicative of terrestrial sources. The fungal biomass was 12- to 100-fold lower than the biomass of pelagic
bacteria, suggesting that fungi in the water column of the rivers probably were of minor importance in the riverine ecosystems
at the sampling time.
[show abstract][hide abstract] ABSTRACT: Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation.
PLoS ONE 01/2012; 7(4):e35314. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
The ISME Journal 09/2011; 6(3):703-7. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C(1) metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean's largest habitat.
[show abstract][hide abstract] ABSTRACT: 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.
The ISME Journal 06/2011; 6(1):113-23. · 8.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Whole-genome shotgun sequence data from three individual cells isolated from seawater, followed by analysis of ribosomal DNA, indicated that the cells represented three divergent clades of picobiliphytes. In contrast with the recent description of this phylum, we found no evidence of plastid DNA nor of nuclear-encoded plastid-targeted proteins, which suggests that these picobiliphytes are heterotrophs. Genome data from one cell were dominated by sequences from a widespread single-stranded DNA virus. This virus was absent from the other two cells, both of which contained non-eukaryote DNA derived from marine Bacteroidetes and large DNA viruses. By using shotgun sequencing of uncultured marine picobiliphytes, we revealed the distinct interactions of individual cells.