2016 |
Y, Zhang; Y, Sun; N, Jiao; R, Stepanauskas; H, Luo Ecological Genomics of the Uncultivated Marine Roseobacter Lineage CHAB-I-5 Journal Article Applied and Environmental Microbiology, 82 (7), 2016. @article{Y2016, title = {Ecological Genomics of the Uncultivated Marine Roseobacter Lineage CHAB-I-5}, author = {Zhang Y and Sun Y and Jiao N and Stepanauskas R and Luo H}, url = {http://aem.asm.org/content/82/7/2100.long}, doi = {10.1128/AEM.03678-15}, year = {2016}, date = {2016-01-29}, journal = {Applied and Environmental Microbiology}, volume = {82}, number = {7}, abstract = {Members of the marine Roseobacter clade are major participants in global carbon and sulfur cycles. While roseobacters are well represented in cultures, several abundant pelagic lineages, including SAG-O19, DC5-80-3, and NAC11-7, remain largely uncultivated and show evidence of genome streamlining. Here, we analyzed the partial genomes of three single cells affiliated with CHAB-I-5, another abundant but exclusively uncultivated Roseobacter lineage. Members of this lineage encode several metabolic potentials that are absent in streamlined genomes. Examples are quorum sensing and type VI secretion systems, which enable them to effectively interact with host and other bacteria. Further analysis of the CHAB-I-5 single-cell amplified genomes (SAGs) predicted that this lineage comprises members with relatively large genomes (4.1 to 4.4 Mbp) and a high fraction of noncoding DNA (10 to 12%), which is similar to what is observed in many cultured, nonstreamlined Roseobacter lineages. The four uncultured lineages, while exhibiting highly variable geographic distributions, together represent >60% of the global pelagic roseobacters. They are consistently enriched in genes encoding the capabilities of light harvesting, oxidation of “energy-rich” reduced sulfur compounds and methylated amines, uptake and catabolism of various carbohydrates and osmolytes, and consumption of abundant exudates from phytoplankton. These traits may define the global prevalence of the four lineages among marine bacterioplankton.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Members of the marine Roseobacter clade are major participants in global carbon and sulfur cycles. While roseobacters are well represented in cultures, several abundant pelagic lineages, including SAG-O19, DC5-80-3, and NAC11-7, remain largely uncultivated and show evidence of genome streamlining. Here, we analyzed the partial genomes of three single cells affiliated with CHAB-I-5, another abundant but exclusively uncultivated Roseobacter lineage. Members of this lineage encode several metabolic potentials that are absent in streamlined genomes. Examples are quorum sensing and type VI secretion systems, which enable them to effectively interact with host and other bacteria. Further analysis of the CHAB-I-5 single-cell amplified genomes (SAGs) predicted that this lineage comprises members with relatively large genomes (4.1 to 4.4 Mbp) and a high fraction of noncoding DNA (10 to 12%), which is similar to what is observed in many cultured, nonstreamlined Roseobacter lineages. The four uncultured lineages, while exhibiting highly variable geographic distributions, together represent >60% of the global pelagic roseobacters. They are consistently enriched in genes encoding the capabilities of light harvesting, oxidation of “energy-rich” reduced sulfur compounds and methylated amines, uptake and catabolism of various carbohydrates and osmolytes, and consumption of abundant exudates from phytoplankton. These traits may define the global prevalence of the four lineages among marine bacterioplankton. |
A, Eiler; R, Mondav; L, Sinclair; L, Fernandez-Vidal; DG, Scofield; P, Schwientek; M, Martinez-Garcia; D, Torrents; KD, McMahon; SGE, Andersson; R, Stepanauskas; T, Woyke; S, Bertilsson Tuning fresh: radiation through rewiring of central metabolism in streamlined bacteria Journal Article ISME, 10 , pp. 1902–1914, 2016. @article{A2016, title = {Tuning fresh: radiation through rewiring of central metabolism in streamlined bacteria}, author = {Eiler A and Mondav R and Sinclair L and Fernandez-Vidal L and Scofield DG and Schwientek P and Martinez-Garcia M and Torrents D and McMahon KD and Andersson SGE and Stepanauskas R and Woyke T and Bertilsson S}, doi = {doi:10.1038/ismej.2015.260}, year = {2016}, date = {2016-01-19}, journal = {ISME}, volume = {10}, pages = {1902\textendash1914}, abstract = {Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner\textendashDoudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner–Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype. |
2015 |
A, Srivastava; KD, McMahon; R, Stepanauskas; HP, Grossart De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany) Journal Article International Microbiology, 19 (39-47), 2015. @article{A2015, title = {De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany)}, author = {Srivastava A and McMahon KD and Stepanauskas R and Grossart HP}, url = {http://dx.doi.org/10.2436/20.1501.01.262}, doi = {10.2436/20.1501.01.262}, year = {2015}, date = {2015-12-18}, journal = {International Microbiology}, volume = {19}, number = {39-47}, abstract = {The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 10 6 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach. Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 10 6 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141-P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoP-annotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. |
DK, Ngugi; J, Blom; R, Stepanauskas; U, Stingl Diversification and niche adaptations of Nitrospina-like bacteria in the polyextreme interfaces of Red Sea brines Journal Article ISME, 10 , pp. 1383–1399, 2015. @article{DK2015, title = {Diversification and niche adaptations of Nitrospina-like bacteria in the polyextreme interfaces of Red Sea brines}, author = {Ngugi DK and Blom J and Stepanauskas R and Stingl U}, url = {https://www.nature.com/articles/ismej2015214}, doi = {10.1038/ismej.2015.214}, year = {2015}, date = {2015-12-15}, journal = {ISME}, volume = {10}, pages = {1383\textendash1399}, abstract = {Nitrite-oxidizing bacteria (NOB) of the genus Nitrospina have exclusively been found in marine environments. In the brine\textendashseawater interface layer of Atlantis II Deep (Red Sea), Nitrospina-like bacteria constitute up to one-third of the bacterial 16S ribosomal RNA (rRNA) gene sequences. This is much higher compared with that reported in other marine habitats (~10% of all bacteria), and was unexpected because no NOB culture has been observed to grow above 4.0% salinity, presumably due to the low net energy gained from their metabolism that is insufficient for both growth and osmoregulation. Using phylogenetics, single-cell genomics and metagenomic fragment recruitment approaches, we document here that these Nitrospina-like bacteria, designated as Candidatus Nitromaritima RS, are not only highly diverged from the type species Nitrospina gracilis (pairwise genome identity of 69%) but are also ubiquitous in the deeper, highly saline interface layers (up to 11.2% salinity) with temperatures of up to 52 °C. Comparative pan-genome analyses revealed that less than half of the predicted proteome of Ca. Nitromaritima RS is shared with N. gracilis. Interestingly, the capacity for nitrite oxidation is also conserved in both genomes. Although both lack acidic proteomes synonymous with extreme halophiles, the pangenome of Ca. Nitromaritima RS specifically encodes enzymes with osmoregulatory and thermoprotective roles (i.e., ectoine/hydroxyectoine biosynthesis) and of thermodynamic importance (i.e., nitrate and nitrite reductases). Ca. Nitromaritima RS also possesses many hallmark traits of microaerophiles and high-affinity NOB. The abundance of the uncultured Ca. Nitromaritima lineage in marine oxyclines suggests their unrecognized ecological significance in deoxygenated areas of the global ocean.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nitrite-oxidizing bacteria (NOB) of the genus Nitrospina have exclusively been found in marine environments. In the brine–seawater interface layer of Atlantis II Deep (Red Sea), Nitrospina-like bacteria constitute up to one-third of the bacterial 16S ribosomal RNA (rRNA) gene sequences. This is much higher compared with that reported in other marine habitats (~10% of all bacteria), and was unexpected because no NOB culture has been observed to grow above 4.0% salinity, presumably due to the low net energy gained from their metabolism that is insufficient for both growth and osmoregulation. Using phylogenetics, single-cell genomics and metagenomic fragment recruitment approaches, we document here that these Nitrospina-like bacteria, designated as Candidatus Nitromaritima RS, are not only highly diverged from the type species Nitrospina gracilis (pairwise genome identity of 69%) but are also ubiquitous in the deeper, highly saline interface layers (up to 11.2% salinity) with temperatures of up to 52 °C. Comparative pan-genome analyses revealed that less than half of the predicted proteome of Ca. Nitromaritima RS is shared with N. gracilis. Interestingly, the capacity for nitrite oxidation is also conserved in both genomes. Although both lack acidic proteomes synonymous with extreme halophiles, the pangenome of Ca. Nitromaritima RS specifically encodes enzymes with osmoregulatory and thermoprotective roles (i.e., ectoine/hydroxyectoine biosynthesis) and of thermodynamic importance (i.e., nitrate and nitrite reductases). Ca. Nitromaritima RS also possesses many hallmark traits of microaerophiles and high-affinity NOB. The abundance of the uncultured Ca. Nitromaritima lineage in marine oxyclines suggests their unrecognized ecological significance in deoxygenated areas of the global ocean. |
JH, Munson-McGee; EK, Field; M, Bateson; C, Rooney; R, Stepanauskas; MJ, Young Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs Journal Article Applied and Environmental Microbiology, 2015. @article{JH2015, title = {Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs}, author = {Munson-McGee JH and Field EK and Bateson M and Rooney C and Stepanauskas R and Young MJ}, url = {http://aem.asm.org/content/81/22/7860.short}, doi = {10.1128/AEM.01539-15}, year = {2015}, date = {2015-09-04}, journal = {Applied and Environmental Microbiology}, abstract = {Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota. |
SA, Carr; BN, Orcutt; KW, Mandernack; JR, Spear Abundant Atribacteria in deep marine sediment from the Adélie Basin, Antarctica Journal Article Frontiers in Microbiology, 2015. @article{SA2015, title = {Abundant Atribacteria in deep marine sediment from the Ad\'{e}lie Basin, Antarctica}, author = {Carr SA and Orcutt BN and Mandernack KW and Spear JR}, url = {https://www.frontiersin.org/articles/10.3389/fmicb.2015.00872/full}, doi = {10.3389/fmicb.2015.00872}, year = {2015}, date = {2015-08-26}, journal = {Frontiers in Microbiology}, abstract = {Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Ad\'{e}lie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment. |
SA, Carr; BN, Orcutt; KW, Mandernack; JR, Spear Abundant Atribacteria in deep marine sediment from the Adélie Basin, Antarctica Journal Article Frontiers in Microbiology, 6 , 2015. @article{SA2015b, title = {Abundant Atribacteria in deep marine sediment from the Ad\'{e}lie Basin, Antarctica}, author = {Carr SA and Orcutt BN and Mandernack KW and Spear JR}, url = {https://www.frontiersin.org/articles/10.3389/fmicb.2015.00872/full}, doi = {10.3389/fmicb.2015.00872}, year = {2015}, date = {2015-08-25}, journal = {Frontiers in Microbiology}, volume = {6}, abstract = {Bacteria belonging to the newly classified candidate phylum "Atribacteria" (formerly referred to as "OP9" and "JS1") are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica's Ad\'{e}lie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Bacteria belonging to the newly classified candidate phylum "Atribacteria" (formerly referred to as "OP9" and "JS1") are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica's Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment. |
JM, Labonté; EK, Field; M, Lau; D, Chivian; van E, Heerden; KE, Wommack; TL, Kieft; TC, Onstott; R, Stepanauskas Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population Journal Article Frontiers in Microbiology, 2015. @article{JM2015b, title = {Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population}, author = {Labont\'{e} JM and Field EK and Lau M and Chivian D and van Heerden E and Wommack KE and Kieft TL and Onstott TC and Stepanauskas R}, url = {https://www.frontiersin.org/articles/10.3389/fmicb.2015.00349/full}, doi = {10.3389/fmicb.2015.00349}, year = {2015}, date = {2015-04-22}, journal = {Frontiers in Microbiology}, abstract = {A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.}, keywords = {}, pubstate = {published}, tppubtype = {article} } A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment. |
JM, Labonté; BK, Swan; B, Poulos; H, Luo; S, Koren; SJ, Hallam; MB, Sullivan; T, Woyke; KE, Wommack; R, Stepanauskas Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton Journal Article ISME, 2015. @article{JM2015, title = {Single-cell genomics-based analysis of virus\textendashhost interactions in marine surface bacterioplankton}, author = {Labont\'{e} JM and Swan BK and Poulos B and Luo H and Koren S and Hallam SJ and Sullivan MB and Woyke T and Wommack KE and Stepanauskas R}, url = {https://www.nature.com/articles/ismej201548}, doi = {10.1038/ismej.2015.48}, year = {2015}, date = {2015-04-17}, journal = {ISME}, abstract = {Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus\textendashhost relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus\textendashhost interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage\textendashhost interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host\textendashvirus interactions in complex microbial communities.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities. |
R, Stepanauskas Wiretapping into microbial interactions by single cell genomics Journal Article Frontiers in Microbiology, 2015. @article{R2015, title = {Wiretapping into microbial interactions by single cell genomics}, author = {Stepanauskas R}, url = {https://www.frontiersin.org/articles/10.3389/fmicb.2015.00258/full}, doi = {10.3389/fmicb.2015.00258}, year = {2015}, date = {2015-04-08}, journal = {Frontiers in Microbiology}, abstract = {Over the past decade, single cell genomics (SCG) made a swift transition from science fiction to a handy new tool in the biologist's toolset. The power of this technology lies in its ability to retrieve information-rich genomic blueprints from the most fundamental units of biological organization\textemdashindividual cells. This is particularly significant in the case of bacteria, archaea, and protists, where single cells constitute complete organisms. Such unicellular individuals comprise the vast majority of biological diversity and biomass of our planet, yet only a small fraction of microbial diversity has been discovered and studied. Together with other modern research tools, SCG has been increasingly instrumental in deciphering the genomic composition, metabolic potential and evolutionary histories of the “microbial dark matter.” While cultivation-free recovery of discrete genomes was impossible in 2004, by 2009 it became a routine procedure that is accessible to the broad research community through open-access SCG core facilities (e.g., scgc.bigelow.org). This rapid development has enabled genomic studies of many previously unexplored branches of the tree of life (Marcy et al., 2007; Rinke et al., 2013) and findings of hitherto unrecognized biogeochemical processes and ecological patterns (Swan et al., 2011, 2013; Mason et al., 2012), paving the way for a new wave of discovery in microbiology and biotechnology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Over the past decade, single cell genomics (SCG) made a swift transition from science fiction to a handy new tool in the biologist's toolset. The power of this technology lies in its ability to retrieve information-rich genomic blueprints from the most fundamental units of biological organization—individual cells. This is particularly significant in the case of bacteria, archaea, and protists, where single cells constitute complete organisms. Such unicellular individuals comprise the vast majority of biological diversity and biomass of our planet, yet only a small fraction of microbial diversity has been discovered and studied. Together with other modern research tools, SCG has been increasingly instrumental in deciphering the genomic composition, metabolic potential and evolutionary histories of the “microbial dark matter.” While cultivation-free recovery of discrete genomes was impossible in 2004, by 2009 it became a routine procedure that is accessible to the broad research community through open-access SCG core facilities (e.g., scgc.bigelow.org). This rapid development has enabled genomic studies of many previously unexplored branches of the tree of life (Marcy et al., 2007; Rinke et al., 2013) and findings of hitherto unrecognized biogeochemical processes and ecological patterns (Swan et al., 2011, 2013; Mason et al., 2012), paving the way for a new wave of discovery in microbiology and biotechnology. |
J, Martijn; F, Schullz; K, Zaremba-Niedzwiedzka; J, Viklund; R, Stepanauskas; SGE, Andersson; M, Horn; L, Guy; TJG, Ettema Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution Journal Article ISME, 2015. @article{J2015, title = {Single-cell genomics of a rare environmental alphaproteobacterium provides unique insights into Rickettsiaceae evolution}, author = {Martijn J and Schullz F and Zaremba-Niedzwiedzka K and Viklund J and Stepanauskas R and Andersson SGE and Horn M and Guy L and Ettema TJG}, url = {https://www.nature.com/articles/ismej201546}, doi = {10.1038/ismej.2015.46}, year = {2015}, date = {2015-04-07}, journal = {ISME}, abstract = {The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris’, a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris’ genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris’, a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris’ genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular. |
Paul BG Bagby SC, Czornyj Arambula Handa Sczyrba Ghosh Miller JF Valentine DL E D S A P Targeted diversity generation by intraterrestrial archaea and archaeal viruses Journal Article Nature Communications, 2015. @article{BG2015, title = {Targeted diversity generation by intraterrestrial archaea and archaeal viruses}, author = {Paul BG, Bagby SC, Czornyj E, Arambula D, Handa S, Sczyrba A, Ghosh P, Miller JF, Valentine DL}, url = {https://www.nature.com/articles/ncomms7585}, doi = {10.1038/ncomms7585}, year = {2015}, date = {2015-03-23}, journal = {Nature Communications}, abstract = {In the evolutionary arms race between microbes, their parasites, and their neighbours, the capacity for rapid protein diversification is a potent weapon. Diversity-generating retroelements (DGRs) use mutagenic reverse transcription and retrohoming to generate myriad variants of a target gene. Originally discovered in pathogens, these retroelements have been identified in bacteria and their viruses, but never in archaea. Here we report the discovery of intact DGRs in two distinct intraterrestrial archaeal systems: a novel virus that appears to infect archaea in the marine subsurface, and, separately, two uncultivated nanoarchaea from the terrestrial subsurface. The viral DGR system targets putative tail fibre ligand-binding domains, potentially generating >1018 protein variants. The two single-cell nanoarchaeal genomes each possess ≥4 distinct DGRs. Against an expected background of low genome-wide mutation rates, these results demonstrate a previously unsuspected potential for rapid, targeted sequence diversification in intraterrestrial archaea and their viruses.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In the evolutionary arms race between microbes, their parasites, and their neighbours, the capacity for rapid protein diversification is a potent weapon. Diversity-generating retroelements (DGRs) use mutagenic reverse transcription and retrohoming to generate myriad variants of a target gene. Originally discovered in pathogens, these retroelements have been identified in bacteria and their viruses, but never in archaea. Here we report the discovery of intact DGRs in two distinct intraterrestrial archaeal systems: a novel virus that appears to infect archaea in the marine subsurface, and, separately, two uncultivated nanoarchaea from the terrestrial subsurface. The viral DGR system targets putative tail fibre ligand-binding domains, potentially generating >1018 protein variants. The two single-cell nanoarchaeal genomes each possess ≥4 distinct DGRs. Against an expected background of low genome-wide mutation rates, these results demonstrate a previously unsuspected potential for rapid, targeted sequence diversification in intraterrestrial archaea and their viruses. |
R, Stepanauskas Crystal Ball: Re-defining microbial diversity from its single-celled building blocks Journal Article Environmental Microbiology Reports, 2015. @article{R2015b, title = {Crystal Ball: Re-defining microbial diversity from its single-celled building blocks}, author = {Stepanauskas R}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/1758-2229.12226}, doi = {10.1111/1758-2229.12226}, year = {2015}, date = {2015-02-26}, journal = {Environmental Microbiology Reports}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
2014 |
EK, Field; A, Sczyrba; AE, Lyman; CC, Harris; T, Woyke; R, Stepanauskas; D, Emerson Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount Journal Article ISME, 2014. @article{EK2014, title = {Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount}, author = {Field EK and Sczyrba A and Lyman AE and Harris CC and Woyke T and Stepanauskas R and Emerson D}, url = {https://www.nature.com/articles/ismej2014183}, doi = {10.1038/ismej.2014.183}, year = {2014}, date = {2014-10-10}, journal = {ISME}, abstract = {The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies. |
M, Martínez-García; F, Santos; M, Moreno-Paz; V, Parro; J, Antón Unveiling viral–host interactions within the ‘microbial dark matter’ Journal Article Nature Communications, 2014. @article{M2014, title = {Unveiling viral\textendashhost interactions within the ‘microbial dark matter’}, author = {Mart\'{i}nez-Garc\'{i}a M and Santos F and Moreno-Paz M and Parro V and Ant\'{o}n J}, url = {https://www.nature.com/articles/ncomms5542}, doi = {10.1038/ncomms5542}, year = {2014}, date = {2014-08-14}, journal = {Nature Communications}, abstract = {Viruses control natural microbial communities. Identification of virus\textendashhost pairs relies either on their cultivation or on metagenomics and tentative assignment based on genomic signatures. Both approaches have severe drawbacks when aiming to target such pairs within the uncultured majority. Here we present an unambiguous way to assign viruses to hosts that does not rely on any previous information about either of them nor requires their cultivation. First, genomic contents of individual cells present in an environmental sample are retrieved by means of single-cell genomic technologies. Then, individual cell genomes are hybridized against a set of individual viral genomes from the same sample, previously immobilized on a microarray. Infected cells will yield positive hybridization as they carry viral genomes, which can be then sequenced and characterized. Using this method, we pinpoint viruses infecting the ubiquitous hyperhalophilic Nanohaloarchaeota, included in the so-called ‘microbial dark matter’ (the uncultured fraction of the microbial world).}, keywords = {}, pubstate = {published}, tppubtype = {article} } Viruses control natural microbial communities. Identification of virus–host pairs relies either on their cultivation or on metagenomics and tentative assignment based on genomic signatures. Both approaches have severe drawbacks when aiming to target such pairs within the uncultured majority. Here we present an unambiguous way to assign viruses to hosts that does not rely on any previous information about either of them nor requires their cultivation. First, genomic contents of individual cells present in an environmental sample are retrieved by means of single-cell genomic technologies. Then, individual cell genomes are hybridized against a set of individual viral genomes from the same sample, previously immobilized on a microarray. Infected cells will yield positive hybridization as they carry viral genomes, which can be then sequenced and characterized. Using this method, we pinpoint viruses infecting the ubiquitous hyperhalophilic Nanohaloarchaeota, included in the so-called ‘microbial dark matter’ (the uncultured fraction of the microbial world). |
DK, Ngugi; J, Blom; I, Alam; M, Rashid; W, Ba-Alawi; G, Zhang; T, Hikmawan; Y, Guan; A, Antunes; R, Siam; H, El Dorry; V, Bajic; U, Stingl Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea Journal Article ISME, 2014. @article{DK2014, title = {Comparative genomics reveals adaptations of a halotolerant thaumarchaeon in the interfaces of brine pools in the Red Sea}, author = {Ngugi DK and Blom J and Alam I and Rashid M and Ba-Alawi W and Zhang G and Hikmawan T and Guan Y and Antunes A and Siam R and El Dorry H and Bajic V and Stingl U}, url = {https://www.nature.com/articles/ismej2014137}, doi = {10.1038/ismej.2014.137}, year = {2014}, date = {2014-08-08}, journal = {ISME}, abstract = {The bottom of the Red Sea harbors over 25 deep hypersaline anoxic basins that are geochemically distinct and characterized by vertical gradients of extreme physicochemical conditions. Because of strong changes in density, particulate and microbial debris get entrapped in the brine-seawater interface (BSI), resulting in increased dissolved organic carbon, reduced dissolved oxygen toward the brines and enhanced microbial activities in the BSI. These features coupled with the deep-sea prevalence of ammonia-oxidizing archaea (AOA) in the global ocean make the BSI a suitable environment for studying the osmotic adaptations and ecology of these important players in the marine nitrogen cycle. Using phylogenomic-based approaches, we show that the local archaeal community of five different BSI habitats (with up to 18.2% salinity) is composed mostly of a single, highly abundant Nitrosopumilus-like phylotype that is phylogenetically distinct from the bathypelagic thaumarchaea; ammonia-oxidizing bacteria were absent. The composite genome of this novel Nitrosopumilus-like subpopulation (RSA3) co-assembled from multiple single-cell amplified genomes (SAGs) from one such BSI habitat further revealed that it shares ∼54% of its predicted genomic inventory with sequenced Nitrosopumilus species. RSA3 also carries several, albeit variable gene sets that further illuminate the phylogenetic diversity and metabolic plasticity of this genus. Specifically, it encodes for a putative proline-glutamate ‘switch’ with a potential role in osmotolerance and indirect impact on carbon and energy flows. Metagenomic fragment recruitment analyses against the composite RSA3 genome, Nitrosopumilus maritimus, and SAGs of mesopelagic thaumarchaea also reiterate the divergence of the BSI genotypes from other AOA.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The bottom of the Red Sea harbors over 25 deep hypersaline anoxic basins that are geochemically distinct and characterized by vertical gradients of extreme physicochemical conditions. Because of strong changes in density, particulate and microbial debris get entrapped in the brine-seawater interface (BSI), resulting in increased dissolved organic carbon, reduced dissolved oxygen toward the brines and enhanced microbial activities in the BSI. These features coupled with the deep-sea prevalence of ammonia-oxidizing archaea (AOA) in the global ocean make the BSI a suitable environment for studying the osmotic adaptations and ecology of these important players in the marine nitrogen cycle. Using phylogenomic-based approaches, we show that the local archaeal community of five different BSI habitats (with up to 18.2% salinity) is composed mostly of a single, highly abundant Nitrosopumilus-like phylotype that is phylogenetically distinct from the bathypelagic thaumarchaea; ammonia-oxidizing bacteria were absent. The composite genome of this novel Nitrosopumilus-like subpopulation (RSA3) co-assembled from multiple single-cell amplified genomes (SAGs) from one such BSI habitat further revealed that it shares ∼54% of its predicted genomic inventory with sequenced Nitrosopumilus species. RSA3 also carries several, albeit variable gene sets that further illuminate the phylogenetic diversity and metabolic plasticity of this genus. Specifically, it encodes for a putative proline-glutamate ‘switch’ with a potential role in osmotolerance and indirect impact on carbon and energy flows. Metagenomic fragment recruitment analyses against the composite RSA3 genome, Nitrosopumilus maritimus, and SAGs of mesopelagic thaumarchaea also reiterate the divergence of the BSI genotypes from other AOA. |
NH, Youssef; C, Rinke; R, Stepanauskas; I, Farag; T, Woyke; MS, Elshahed Insights into the metabolism, lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites’ Journal Article ISME, 2014. @article{NH2014, title = {Insights into the metabolism, lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites’}, author = {Youssef NH and Rinke C and Stepanauskas R and Farag I and Woyke T and Elshahed MS}, url = {https://www.nature.com/articles/ismej2014141}, doi = {10.1038/ismej.2014.141}, year = {2014}, date = {2014-08-01}, journal = {ISME}, abstract = {The archaeal phylum ‘Diapherotrites’ was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the ‘Diapherotrites’. The most complete of the SAGs, Candidatus ‘Iainarchaeum andersonii’ (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the ‘Diapherotrites’ have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. ‘Diapherotrites’ 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The archaeal phylum ‘Diapherotrites’ was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the ‘Diapherotrites’. The most complete of the SAGs, Candidatus ‘Iainarchaeum andersonii’ (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the ‘Diapherotrites’ have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. ‘Diapherotrites’ 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation. |
M, Gomariz; M, Martinez-Garcia; F, Santos; F, Rodriguez; S, Capella-Gutierrez; T, Gabaldon; R, Rosello-Mora From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists Journal Article ISME, 2014. @article{M2014b, title = {From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists}, author = {Gomariz M and Martinez-Garcia M and Santos F and Rodriguez F and Capella-Gutierrez S and Gabaldon T and Rosello-Mora R}, url = {https://www.nature.com/articles/ismej201495}, doi = {10.1038/ismej.2014.95}, year = {2014}, date = {2014-06-13}, journal = {ISME}, abstract = {The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group. |
Kashtan, Nadav; Roggensack, Sara E; Rodrigue, Sébastien; Thompson, Jessie W; Biller, Steven J; Coe, Allison; Ding, Huiming; Marttinen, Pekka; Malmstrom, Rex R; Stocker, Roman; Follows, Michael J; Stepanauskas, Ramunas; Chisholm, Sallie W Single-Cell Genomics Reveals Hundreds of Coexisting Subpopulations in Wild Prochlorococcus Journal Article Science, 344 (6182), pp. 416-420, 2014. @article{Kashtan12014, title = {Single-Cell Genomics Reveals Hundreds of Coexisting Subpopulations in Wild Prochlorococcus}, author = {Nadav Kashtan and Sara E Roggensack and S\'{e}bastien Rodrigue and Jessie W Thompson and Steven J Biller and Allison Coe and Huiming Ding and Pekka Marttinen and Rex R Malmstrom and Roman Stocker and Michael J Follows and Ramunas Stepanauskas and Sallie W Chisholm}, url = {http://science.sciencemag.org/content/344/6182/416}, doi = {10.1126/science.1248575}, year = {2014}, date = {2014-04-25}, journal = {Science}, volume = {344}, number = {6182}, pages = {416-420}, abstract = {Extensive genomic diversity within coexisting members of a microbial species has been revealed through selected cultured isolates and metagenomic assemblies. Yet, the cell-by-cell genomic composition of wild uncultured populations of co-occurring cells is largely unknown. In this work, we applied large-scale single-cell genomics to study populations of the globally abundant marine cyanobacterium Prochlorococcus. We show that they are composed of hundreds of subpopulations with distinct “genomic backbones,” each backbone consisting of a different set of core gene alleles linked to a small distinctive set of flexible genes. These subpopulations are estimated to have diverged at least a few million years ago, suggesting ancient, stable niche partitioning. Such a large set of coexisting subpopulations may be a general feature of free-living bacterial species with huge populations in highly mixed habitats.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Extensive genomic diversity within coexisting members of a microbial species has been revealed through selected cultured isolates and metagenomic assemblies. Yet, the cell-by-cell genomic composition of wild uncultured populations of co-occurring cells is largely unknown. In this work, we applied large-scale single-cell genomics to study populations of the globally abundant marine cyanobacterium Prochlorococcus. We show that they are composed of hundreds of subpopulations with distinct “genomic backbones,” each backbone consisting of a different set of core gene alleles linked to a small distinctive set of flexible genes. These subpopulations are estimated to have diverged at least a few million years ago, suggesting ancient, stable niche partitioning. Such a large set of coexisting subpopulations may be a general feature of free-living bacterial species with huge populations in highly mixed habitats. |
LJ, Baker; PF, Kemp Exploring bacteria–diatom associations using single-cell whole genome amplification Journal Article Aquatic Microbial Ecology, 72 , pp. 73-88, 2014. @article{LJ2014, title = {Exploring bacteria\textendashdiatom associations using single-cell whole genome amplification}, author = {Baker LJ and Kemp PF}, url = {http://www.int-res.com/abstracts/ame/v72/n1/p73-88/}, doi = {10.3354/ame01686}, year = {2014}, date = {2014-04-04}, journal = {Aquatic Microbial Ecology}, volume = {72}, pages = {73-88}, abstract = {Diatoms are responsible for a large fraction of oceanic and freshwater biomass production and are critically important for sequestration of carbon to the deep ocean. As with most surfaces present in aquatic systems, bacteria colonize the exterior of diatom cells, and they interact with the diatom and each other. The ecology of diatoms may be better explained by conceptualizing them as composite organisms consisting of the host cell and its bacterial associates. Such associations could have collective properties that are not predictable from the properties of the host cell alone. Past studies of these associations have employed culture-based, whole-population methods. In contrast, we examined the composition and variability of bacterial assemblages attached to individual diatoms. Samples were collected in an oligotrophic system (Station ALOHA, 22°45’N, 158°00’W) at the deep chlorophyll maximum. Forty eukaryotic host cells were isolated by flow cytometry followed by multiple displacement amplification, including 26 Thalassiosira spp., other diatoms, dinoflagellates, coccolithophorids, and flagellates. Bacteria were identified by amplifying, cloning, and sequencing 16S rDNA using primers that select against chloroplast 16S rDNA. Bacterial sequences were recovered from 32 of 40 host cells, and from parallel samples of the free-living and particle-associated bacteria. Bacterial assemblages varied substantially even among closely related host cells. Host cells and the free-living and particle-associated samples can be placed into distinct groups based on the phylogenetic relatedness of their associated bacteria, rather than the identity of the host cell. As yet, the functional implications of these groups are unknown.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Diatoms are responsible for a large fraction of oceanic and freshwater biomass production and are critically important for sequestration of carbon to the deep ocean. As with most surfaces present in aquatic systems, bacteria colonize the exterior of diatom cells, and they interact with the diatom and each other. The ecology of diatoms may be better explained by conceptualizing them as composite organisms consisting of the host cell and its bacterial associates. Such associations could have collective properties that are not predictable from the properties of the host cell alone. Past studies of these associations have employed culture-based, whole-population methods. In contrast, we examined the composition and variability of bacterial assemblages attached to individual diatoms. Samples were collected in an oligotrophic system (Station ALOHA, 22°45’N, 158°00’W) at the deep chlorophyll maximum. Forty eukaryotic host cells were isolated by flow cytometry followed by multiple displacement amplification, including 26 Thalassiosira spp., other diatoms, dinoflagellates, coccolithophorids, and flagellates. Bacteria were identified by amplifying, cloning, and sequencing 16S rDNA using primers that select against chloroplast 16S rDNA. Bacterial sequences were recovered from 32 of 40 host cells, and from parallel samples of the free-living and particle-associated bacteria. Bacterial assemblages varied substantially even among closely related host cells. Host cells and the free-living and particle-associated samples can be placed into distinct groups based on the phylogenetic relatedness of their associated bacteria, rather than the identity of the host cell. As yet, the functional implications of these groups are unknown. |
2013 |
K, Zaremba-Niedzwiedzka; J, Viklund; W, Zhao; J, Ast; A, Sczyrba; T, Woyke; KD, McMahon; S, Bertilsson; R, Stepanauskas; SGE, Andersson Single cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade Journal Article Genome Biology, 14 (R130), 2013, ISSN: 1474-760X. @article{K2013, title = {Single cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade}, author = {Zaremba-Niedzwiedzka K and Viklund J and Zhao W and Ast J and Sczyrba A and Woyke T and McMahon KD and Bertilsson S and Stepanauskas R and Andersson SGE}, issn = {1474-760X}, year = {2013}, date = {2013-11-28}, journal = {Genome Biology}, volume = {14}, number = {R130}, abstract = {Background 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. Results 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. Conclusions 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Background 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. Results 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. Conclusions 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. |
S, Nurk; A, Bankevich; D, Antipov; AA, Gurevich; A, Korobeynikov; A, Lapidus; AD, Prjibelski; A, Pyshkin; A, Sirotkin; Y, Sirotkin; R, Stepanauskas; SR, Clingenpeel; T, Woyke; JS, McLean; R, Lasken; G, Tesler; MA, Alekseyev; PA, Pevzner Assembling single-cell genomes and mini-metagenomes from chimeric MDA products Journal Article Journal of Computational Biology, 20 , pp. 714-737, 2013. @article{S2013, title = {Assembling single-cell genomes and mini-metagenomes from chimeric MDA products}, author = {Nurk S and Bankevich A and Antipov D and Gurevich AA and Korobeynikov A and Lapidus A and Prjibelski AD and Pyshkin A and Sirotkin A and Sirotkin Y and Stepanauskas R and Clingenpeel SR and Woyke T and McLean JS and Lasken R and Tesler G and Alekseyev MA and Pevzner PA}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791033/}, doi = {10.1089/cmb.2013.0084}, year = {2013}, date = {2013-10-20}, journal = {Journal of Computational Biology}, volume = {20}, pages = {714-737}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
GM, Garrity; J, Banfield; van der N, Eisen Lelie J; T, McMahon; D, Rusch; E, Delong; MA, Moran; C, Currie; J, Furhman; S, Hallam; P, Hugenholtz; N, Moran; K, Nelson; R, Roberts; R, Stepanauskas Prokaryotic super program advisory committee DOE joint genome institute, Walnut Creek, CA, March 27, 2012 Journal Article Standards in Genomic Sciences, 8 (3), pp. 561-570, 2013. @article{GM2013, title = {Prokaryotic super program advisory committee DOE joint genome institute, Walnut Creek, CA, March 27, 2012}, author = {Garrity GM and Banfield J and Eisen J van der Lelie N and McMahon T and Rusch D and Delong E and Moran MA and Currie C and Furhman J and Hallam S and Hugenholtz P and Moran N and Nelson K and Roberts R and Stepanauskas R}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910701/}, doi = {10.4056/sigs.4638348}, year = {2013}, date = {2013-07-30}, journal = {Standards in Genomic Sciences}, volume = {8}, number = {3}, pages = {561-570}, abstract = {The Prokaryotic Super Program Advisory Committee met on March 27, 2013 for their annual review the Prokaryotic Super Program at the DOE Joint Genome Institute. As is the case with any site visit or program review, the objective is to evaluate progress in meeting organizational objectives, provide feedback to from the user-community and to assist the JGI in formulating plans for the coming year. The advisors want to commend the JGI for its central role in developing new technologies and capabilities, and for catalyzing the formation of new collaborative user communities. Highlights of the post-meeting exchanges among the advisors focused on the importance of programmatic initiatives including: • GEBA, which serves as a phylogenetic “base-map” on which our knowledge of functional diversity can be layered. • FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions. • Single-cell genomics technology, which is seen to significantly enhance our ability to interpret genomic and metagenomic data and broaden the scope of the GEBA program to encompass at least a part of the microbial “dark-matter”. • IMG, which is seen to play a central role in JGI programs and is viewed as a strategically important asset in the JGI portfolio. On this latter point, the committee encourages the formation of a strategic relationship between IMG and the Kbase to ensure that the intelligence, deep knowledge and experience captured in the former is not lost. The committee strongly urges the DOE to continue its support for maintaining this critical resource.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Prokaryotic Super Program Advisory Committee met on March 27, 2013 for their annual review the Prokaryotic Super Program at the DOE Joint Genome Institute. As is the case with any site visit or program review, the objective is to evaluate progress in meeting organizational objectives, provide feedback to from the user-community and to assist the JGI in formulating plans for the coming year. The advisors want to commend the JGI for its central role in developing new technologies and capabilities, and for catalyzing the formation of new collaborative user communities. Highlights of the post-meeting exchanges among the advisors focused on the importance of programmatic initiatives including: • GEBA, which serves as a phylogenetic “base-map” on which our knowledge of functional diversity can be layered. • FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions. • Single-cell genomics technology, which is seen to significantly enhance our ability to interpret genomic and metagenomic data and broaden the scope of the GEBA program to encompass at least a part of the microbial “dark-matter”. • IMG, which is seen to play a central role in JGI programs and is viewed as a strategically important asset in the JGI portfolio. On this latter point, the committee encourages the formation of a strategic relationship between IMG and the Kbase to ensure that the intelligence, deep knowledge and experience captured in the former is not lost. The committee strongly urges the DOE to continue its support for maintaining this critical resource. |
C, Rinke; P, Schwientek; A, Sczyrba; NN, Ivanova; IJ, Anderson; J-F, Cheng; A, Darling; S, Malfatti; BK, Swan; EA, Gies; JA, Dodsworth; BP, Hedlund; G, Tsiamis; SM, Sievert; W-T, Liu; JA, Eisen; S, Hallam; N, Kyrpides; R, Stepanauskas; E, Rubin; P, Hugenholtz; T, Woyke Insights into the phylogeny and coding potential of microbial dark matter Journal Article Nature, 499 , pp. 431-437, 2013. @article{C2013, title = {Insights into the phylogeny and coding potential of microbial dark matter}, author = {Rinke C and Schwientek P and Sczyrba A and Ivanova NN and Anderson IJ and Cheng J-F and Darling A and Malfatti S and Swan BK and Gies EA and Dodsworth JA and Hedlund BP and Tsiamis G and Sievert SM and Liu W-T and Eisen JA and Hallam S and Kyrpides N and Stepanauskas R and Rubin E and Hugenholtz P and Woyke T}, url = {https://www.nature.com/articles/nature12352}, doi = {10.1038/nature12352}, year = {2013}, date = {2013-07-14}, journal = {Nature}, volume = {499}, pages = {431-437}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
BK, Swan; B, Tupper; A, Sczyrba; FM, Lauro; M, Martinez-Garcia; JM, Gonzalez; H, Luo; JJ, Wright; ZC, Landry; NW, Hanson; B, Thompson; NJ, Poulton; P, Schwientek; S, Gonzalez-Acinas; SJ, Giovannoni; MA, Moran; SJ, Hallam; R, Cavicchioli; T, Woyke; R, Stepanauskas Prevalent genome streamlining and latitudinal divergence of marine bacteria in the surface ocean Journal Article PNAS, 110 (28), pp. 11463-11468, 2013. @article{BK2013, title = {Prevalent genome streamlining and latitudinal divergence of marine bacteria in the surface ocean}, author = {Swan BK and Tupper B and Sczyrba A and Lauro FM and Martinez-Garcia M and Gonzalez JM and Luo H and Wright JJ and Landry ZC and Hanson NW and Thompson B and Poulton NJ and Schwientek P and Gonzalez-Acinas S and Giovannoni SJ and Moran MA and Hallam SJ and Cavicchioli R and Woyke T and Stepanauskas R}, url = {http://www.pnas.org/content/110/28/11463}, doi = {https://doi.org/10.1073/pnas.1304246110}, year = {2013}, date = {2013-07-09}, journal = {PNAS}, volume = {110}, number = {28}, pages = {11463-11468}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
KG, Lloyd; L, Schreiber; DG, Petersen; K, Kjeldsen; MA, Lever; R, Stepanauskas; M, Richter; S, Kleindienst; S, Lenk; A, Schramm; BB, Jorgensen Predominant archaea in marine sediments degrade detrital proteins Journal Article Nature, 496 , pp. 215-182, 2013. @article{KG2013, title = {Predominant archaea in marine sediments degrade detrital proteins}, author = {Lloyd KG and Schreiber L and Petersen DG and Kjeldsen K and Lever MA and Stepanauskas R and Richter M and Kleindienst S and Lenk S and Schramm A and Jorgensen BB}, url = {https://www.nature.com/articles/nature12033}, doi = {10.1038/nature12033}, year = {2013}, date = {2013-04-11}, journal = {Nature}, volume = {496}, pages = {215-182}, abstract = {Half of the microbial cells in the Earth’s oceans are found in sediments1. Many of these cells are members of the Archaea2, 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 Thaumarchaeota3 and Aigarchaeota4, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Half of the microbial cells in the Earth’s oceans are found in sediments1. Many of these cells are members of the Archaea2, 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 Thaumarchaeota3 and Aigarchaeota4, 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. |
O, Zhaxybayeva; R, Stepanauskas; NR, Mohan; RT, Papke Cell sorting analysis of geographically separated hypersaline environments Journal Article Extremophiles, 17 (2), pp. 265-75, 2013. @article{O2013, title = {Cell sorting analysis of geographically separated hypersaline environments}, author = {Zhaxybayeva O and Stepanauskas R and Mohan NR and Papke RT}, url = {https://www.ncbi.nlm.nih.gov/pubmed/23358730}, doi = {10.1007/s00792-013-0514-z}, year = {2013}, date = {2013-03-17}, journal = {Extremophiles}, volume = {17}, number = {2}, pages = {265-75}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
2012 |
Stepanauskas, R Single cell genomics: an individual look at microbes Journal Article Current Opinion in Microbiology, 15 , pp. 613-620, 2012. @article{Stepanauskas2012, title = {Single cell genomics: an individual look at microbes}, author = {Stepanauskas, R}, url = {https://www.sciencedirect.com/science/article/pii/S1369527412001166}, doi = {https://doi.org/10.1016/j.mib.2012.09.001}, year = {2012}, date = {2012-09-29}, journal = {Current Opinion in Microbiology}, volume = {15}, pages = {613-620}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
LG, Garcia; KD, McMahon; M, Martinez-Garcia; A, Srivastava; A, Sczyrba; R, Stepanauskas; HP, Grossart; T, Woyke; F, Warnecke Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton Journal Article The ISME Journal, 7 , pp. 137-147, 2012. @article{LG2012, title = {Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton}, author = {Garcia LG and McMahon KD and Martinez-Garcia M and Srivastava A and Sczyrba A and Stepanauskas R and Grossart HP and Woyke T and Warnecke F}, url = {https://www.nature.com/articles/ismej201286}, doi = {10.1038/ismej.2012.86}, year = {2012}, date = {2012-07-19}, journal = {The ISME Journal}, volume = {7}, pages = {137-147}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
M, Martinez-Garcia; DM, Brazel; BK, Swan; C, Arnosti; PSG, Chain; KG, Reitenga; G, Xie; NJ, Poulton; M, Lluesma Gomez; DED, Masland; B, Thompson; WK, Bellows; K, Ziervogel; CC, Lo; S, Ahmed; CD, Gleasner; CJ, Detter; R, Stepanauskas Capturing single cell genomes of active polysaccharide degraders: An unexpected contribution of Verrucomicrobia Journal Article PLoS ONE, 7 (4), pp. e35314, 2012. @article{M2012, title = {Capturing single cell genomes of active polysaccharide degraders: An unexpected contribution of Verrucomicrobia}, author = {Martinez-Garcia M and Brazel DM and Swan BK and Arnosti C and Chain PSG and Reitenga KG and Xie G and Poulton NJ and Lluesma Gomez M and Masland DED and Thompson B and Bellows WK and Ziervogel K and Lo CC and Ahmed S and Gleasner CD and Detter CJ and Stepanauskas R}, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035314}, doi = {https://doi.org/10.1371/journal.pone.0035314}, year = {2012}, date = {2012-04-20}, journal = {PLoS ONE}, volume = {7}, number = {4}, pages = {e35314}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
2011 |
R, Ghai; L, Pasic; AB, Fernandez; AB, Martin-Cuadrado; CM, Mizuno; KD, McMahon; RT, Papke; R, Stepanauskas; B, Rodriguez-Brito; F, Rohwer; C, Sanchez-Porro; A, Ventosa; F, Rodriguez-Valera New abundant microbial groups in aquatic hypersaline environments Journal Article Scientific Report, 1 (135), 2011. @article{R2011, title = {New abundant microbial groups in aquatic hypersaline environments}, author = {Ghai R and Pasic L and Fernandez AB and Martin-Cuadrado AB and Mizuno CM and McMahon KD and Papke RT and Stepanauskas R and Rodriguez-Brito B and Rohwer F and Sanchez-Porro C and Ventosa A and Rodriguez-Valera F}, url = {https://www.nature.com/articles/srep00135}, doi = {10.1038/srep00135}, year = {2011}, date = {2011-10-31}, journal = {Scientific Report}, volume = {1}, number = {135}, abstract = {We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach. |
T, Woyke; A, Sczyrba; J, Lee; C, Rinke; D, Tighe; S, Clingenpeel; R, Malmstrom; R, Stepanauskas; J-F, Cheng Decontamination of MDA Reagents for Single Cell Whole Genome Amplification Journal Article PLoS ONE, 6 (10), pp. e26161, 2011. @article{T2011, title = {Decontamination of MDA Reagents for Single Cell Whole Genome Amplification}, author = {Woyke T and Sczyrba A and Lee J and Rinke C and Tighe D and Clingenpeel S and Malmstrom R and Stepanauskas R and Cheng J-F}, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026161}, doi = {https://doi.org/10.1371/journal.pone.0026161}, year = {2011}, date = {2011-10-20}, journal = {PLoS ONE}, volume = {6}, number = {10}, pages = {e26161}, abstract = {Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells. |
M, Martinez-Garcia; DM, Brazel; N, Poulton; BK, Swan; M, Lluesma Gomez; D, Masland; ME, Sieracki; R, Stepanauskas Unveiling in situ interactions between marine protists and bacteria through single cell sequencing Journal Article The ISME Journal, 6 , pp. 703-707, 2011. @article{M2011, title = {Unveiling in situ interactions between marine protists and bacteria through single cell sequencing}, author = {Martinez-Garcia M and Brazel DM and Poulton N and Swan BK and Lluesma Gomez M and Masland D and Sieracki ME and Stepanauskas R}, url = {https://www.nature.com/articles/ismej2011126}, doi = {10.1038/ismej.2011.126}, year = {2011}, date = {2011-09-22}, journal = {The ISME Journal}, volume = {6}, pages = {703-707}, 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\textendashprey 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
Engel P, ; R, Stepanauskas; A, Moran N Hidden Diversity in Honey Bee Gut Symbionts Detected by Single-Cell Genomics Journal Article PLOS Genetics, 10 (9), pp. 1004596, 2011. @article{Engel P2011, title = {Hidden Diversity in Honey Bee Gut Symbionts Detected by Single-Cell Genomics}, author = {Engel P and Stepanauskas R and Moran N A}, url = {https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004596}, doi = {10.1371/journal.pgen.1004596}, year = {2011}, date = {2011-09-11}, journal = {PLOS Genetics}, volume = {10}, number = {9}, pages = {1004596}, abstract = {Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification. |
BK, Swan; M, Martinez-Garcia; CM, Preston; A, Sczyrba; T, Woyke; D, Lamy; T, Reinthaler; NJ, Poulton; D, Masland; M, Lluesma Gomez; ME, Sieracki; EF, DeLong; GJ, Herndl; R, Stepanauskas Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean Journal Article Science, 333 (6047), pp. 1296-1300, 2011. @article{BK2011, title = {Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean}, author = {Swan BK and Martinez-Garcia M and Preston CM and Sczyrba A and Woyke T and Lamy D and Reinthaler T and Poulton NJ and Masland D and Lluesma Gomez M and Sieracki ME and DeLong EF and Herndl GJ and Stepanauskas R}, url = {http://science.sciencemag.org/content/333/6047/1296}, doi = {10.1126/science.1203690}, year = {2011}, date = {2011-09-02}, journal = {Science}, volume = {333}, number = {6047}, pages = {1296-1300}, 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 C1 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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 C1 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. |
J, Martinez-Martinez; N, Poulton; R, Stepanauskas; M, Sieracki; WH, Wilson Targeted Sorting of Single Virus-Infected Cells of the Coccolithophore Emiliania huxleyi Journal Article PLoS ONE, 6 (7), pp. e22520, 2011. @article{J2011, title = {Targeted Sorting of Single Virus-Infected Cells of the Coccolithophore Emiliania huxleyi}, author = {Martinez-Martinez J and Poulton N and Stepanauskas R and Sieracki M and Wilson WH }, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022520}, doi = {https://doi.org/10.1371/journal.pone.0022520}, year = {2011}, date = {2011-07-26}, journal = {PLoS ONE}, volume = {6}, number = {7}, pages = {e22520}, abstract = {Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems. |
M, Martinez-Garcia; BK, Swan; NJ, Poulton; M, Lluesma Gomez; D, Masland; ME, Sieracki; R, Stepanauskas High throughput single cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton Journal Article The ISME Journal, 6 , pp. 113-123, 2011. @article{M2011b, title = {High throughput single cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton}, author = {Martinez-Garcia M and Swan BK and Poulton NJ and Lluesma Gomez M and Masland D and Sieracki ME and Stepanauskas R}, url = {https://www.nature.com/articles/ismej201184}, doi = {10.1038/ismej.2011.84}, year = {2011}, date = {2011-06-30}, journal = {The ISME Journal}, volume = {6}, pages = {113-123}, 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\textendash23% 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
HS, Yoon; DC, Price; R, Stepanauskas; VD, Rajah; ME, Sieracki; WH, Wilson; EC, Yang; S, Duffy; D, Bhattacharya Single-cell genomics reveals organismal interactions in uncultivated marine protists Journal Article Science, 332 (6030), pp. 714-717, 2011. @article{HS2011, title = {Single-cell genomics reveals organismal interactions in uncultivated marine protists}, author = {Yoon HS and Price DC and Stepanauskas R and Rajah VD and Sieracki ME and Wilson WH and Yang EC and Duffy S and Bhattacharya D}, url = {http://science.sciencemag.org/content/332/6030/714.long}, doi = {10.1126/science.1203163}, year = {2011}, date = {2011-05-06}, journal = {Science}, volume = {332}, number = {6030}, pages = {714-717}, 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.}, keywords = {}, pubstate = {published}, tppubtype = {article} } 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. |
EJ, Fleming; AE, Langdon; M, Martinez-Garcia; R, Stepanauskas; N, Poulton; D, Masland; D, Emerson What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH Journal Article PLoS ONE, 6 (3), pp. e17769, 2011. @article{EJ2011, title = {What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH}, author = {Fleming EJ and Langdon AE and Martinez-Garcia M and Stepanauskas R and Poulton N and Masland D and Emerson D}, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017769}, doi = {https://doi.org/10.1371/journal.pone.0017769}, year = {2011}, date = {2011-03-17}, journal = {PLoS ONE}, volume = {6}, number = {3}, pages = {e17769}, abstract = {Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms. |
M, Hess; A, Sczyrba; R, Egan; TW, Kim; H, Chokhawala; G, Schroth; S, Luo; DS, Clark; F, Chen; T, Zhang; RI, Mackie; LA, Pennacchio; SG, Tringe; A, Visel; T, Woyke; Z, Wang; EM, Rubin Metagenomic discovery of biomass-degrading genes and genomes from cow rumen Journal Article Science, 331 (6016), pp. 463-467, 2011. @article{M2011c, title = {Metagenomic discovery of biomass-degrading genes and genomes from cow rumen}, author = {Hess M and Sczyrba A and Egan R and Kim TW and Chokhawala H and Schroth G and Luo S and Clark DS and Chen F and Zhang T and Mackie RI and Pennacchio LA and Tringe SG and Visel A and Woyke T and Wang Z and Rubin EM}, url = {http://science.sciencemag.org/content/331/6016/463.long}, doi = {10.1126/science.1200387}, year = {2011}, date = {2011-01-28}, journal = {Science}, volume = {331}, number = {6016}, pages = {463-467}, abstract = {The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass. |
2010 |
JL, Heywood; ME, Sieracki; W, Bellows; NJ, Poulton; R, Stepanauskas Capturing diversity of marine heterotrophic protists: one cell at a time Journal Article The ISME Journal, 5 , pp. 674-684, 2010. @article{JL2010, title = {Capturing diversity of marine heterotrophic protists: one cell at a time}, author = {Heywood JL and Sieracki ME and Bellows W and Poulton NJ and Stepanauskas R}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105736/}, doi = {10.1038/ismej.2010.155}, year = {2010}, date = {2010-10-20}, journal = {The ISME Journal}, volume = {5}, pages = {674-684}, abstract = {Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (o10 lm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent applications of culture-independent, molecular methods have revealed unexpectedly high diversity in a variety of functional and phylogenetic groups of microorganisms in the ocean. However, none of the existing research tools are free from significant limitations, such as PCR and cloning biases, low phylogenetic resolution and others. Here, we employed novel, single-cell sequencing techniques to assess the composition of small (o10 lm diameter), heterotrophic protists from the Gulf of Maine. Single cells were isolated by flow cytometry, their genomes amplified, and 18S rRNA marker genes were amplified and sequenced. We compared the results to traditional environmental PCR cloning of sorted cells. The diversity of heterotrophic protists was significantly higher in the library of single amplified genomes (SAGs) than in environmental PCR clone libraries of the 18S rRNA gene, obtained from the same coastal sample. Libraries of SAGs, but not clones contained several recently discovered, uncultured groups, including picobiliphytes and novel marine stramenopiles. Clone, but not SAG, libraries contained several large clusters of identical and nearly identical sequences of Dinophyceae, Cercozoa and Stramenopiles. Similar results were obtained using two alternative primer sets, suggesting that PCR biases may not be the only explanation for the observed patterns. Instead, differences in the number of 18S rRNA gene copies among the various protist taxa probably had a significant role in determining the PCR clone composition. These results show that single-cell sequencing has the potential to more accurately assess protistan community composition than previously established methods. In addition, the creation of SAG libraries opens opportunities for the analysis of multiple genes or entire genomes of the uncultured protist groups |
A, Reyes-Prieto; HS, Yoon; A, Moustafa; EC, Yang; RA, Andersen; SM, Boo; T, Nakayama; KI, Ishida; D, Bhattacharya Differential gene retention in plastids of common recent origin Journal Article Molecular biology and evolution, 27 (7), pp. 1530-1537, 2010. @article{A2010, title = {Differential gene retention in plastids of common recent origin}, author = {Reyes-Prieto A and Yoon HS and Moustafa A and Yang EC and Andersen RA and Boo SM and Nakayama T and Ishida KI and Bhattacharya D}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912470/}, doi = {10.1093/molbev/msq032}, year = {2010}, date = {2010-02-01}, journal = {Molecular biology and evolution}, volume = {27}, number = {7}, pages = {1530-1537}, abstract = {The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote\textendasheukaryote (primary) endosymbiosis that occurred approximately 60 mya. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence with the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus\textendashProchlorococcus group, and the cyanobacterial provenance of all plastid genes suggests that these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/ dS ratios ,,1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote–eukaryote (primary) endosymbiosis that occurred approximately 60 mya. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence with the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus–Prochlorococcus group, and the cyanobacterial provenance of all plastid genes suggests that these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/ dS ratios ,,1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution. |
2009 |
T, Woyke; G, Xie; A, Copeland; JM, Gonzalez; C, Han; H, Kiss; J, Saw; P, Senin; C, Yang; S, Chatterji; J-F, Cheng; JA, Eisen; ME, Sieracki; R, Stepanauskas Assembling the marine metagenome, one cell at a time Journal Article PLoS ONE, 4 (4), pp. e5299, 2009. @article{T2009, title = {Assembling the marine metagenome, one cell at a time}, author = {Woyke T and Xie G and Copeland A and Gonzalez JM and Han C and Kiss H and Saw J and Senin P and Yang C and Chatterji S and Cheng J-F and Eisen JA and Sieracki ME and Stepanauskas R}, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005299}, doi = {https://doi.org/10.1371/journal.pone.0005299}, year = {2009}, date = {2009-04-23}, journal = {PLoS ONE}, volume = {4}, number = {4}, pages = {e5299}, abstract = {The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa. |
2008 |
T, Ishoey; T, Woyke; R, Stepanauskas; M, Novotny; RS, Lasken Genomic sequencing of single microbial cells from environmental samples Journal Article Current Opinion in Microbiology, 11 (3), pp. 198-204, 2008. @article{T2008, title = {Genomic sequencing of single microbial cells from environmental samples}, author = {Ishoey T and Woyke T and Stepanauskas R and Novotny M and Lasken RS}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3635501/}, doi = {10.1016/j.mib.2008.05.006}, year = {2008}, date = {2008-06-01}, journal = {Current Opinion in Microbiology}, volume = {11}, number = {3}, pages = {198-204}, abstract = {Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing. |
2007 |
R, Stepanauskas; ME, Sieracki Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time Journal Article PNAS, 104 (21), pp. 9052-9057, 2007. @article{R2007, title = {Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time}, author = {Stepanauskas R and Sieracki ME}, url = {http://www.pnas.org/content/104/21/9052.long}, doi = {https://doi.org/10.1073/pnas.0700496104}, year = {2007}, date = {2007-05-22}, journal = {PNAS}, volume = {104}, number = {21}, pages = {9052-9057}, abstract = {The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacteria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88\textendash97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted <1% of the sampled bacterioplankton. Compared with metagenomics, the power of our approach lies in the ability to detect metabolic genes in uncultured microorganisms directly, even when the metabolic and phylogenetic markers are located far apart on the chromosome.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacteria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88–97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted <1% of the sampled bacterioplankton. Compared with metagenomics, the power of our approach lies in the ability to detect metabolic genes in uncultured microorganisms directly, even when the metabolic and phylogenetic markers are located far apart on the chromosome. |
