2014
Kashtan, Nadav; Roggensack, Sara E; Rodrigue, SĂ©bastien; 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
In: Science, vol. 344, no. 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 âgenomic backbones,â 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 âgenomic backbones,â 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 bacteriaâdiatom associations using single-cell whole genome amplification Journal Article
In: Aquatic Microbial Ecology, vol. 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°45âN, 158°00âW) 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°45âN, 158°00âW) 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
In: Genome Biology, vol. 14, no. 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.
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
In: Journal of Computational Biology, vol. 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 âmicrobial dark matterâ 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 âmicrobial dark matterâ 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 Lelie N, Eisen 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
In: Standards in Genomic Sciences, vol. 8, no. 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:
âą GEBA, which serves as a phylogenetic âbase-mapâ on which our knowledge of functional diversity can be layered.
âą FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions.
âą 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 âdark-matterâ.
âą 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:
âą GEBA, which serves as a phylogenetic âbase-mapâ on which our knowledge of functional diversity can be layered.
âą FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions.
âą 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 âdark-matterâ.
âą 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.
âą GEBA, which serves as a phylogenetic âbase-mapâ on which our knowledge of functional diversity can be layered.
âą FEBA, which promises to provide new insights into the physiological capabilities of prokaryotes under highly standardized conditions.
âą 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 âdark-matterâ.
âą 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
In: Nature, vol. 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 201âuncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29âmajor mostly uncharted branches of the tree of life, so-called âmicrobial dark matterâ. 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 201âuncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29âmajor mostly uncharted branches of the tree of life, so-called âmicrobial dark matterâ. 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
In: PNAS, vol. 110, no. 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
In: Nature, vol. 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 Earthâs 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 Earthâs 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
In: Extremophiles, vol. 17, no. 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
In: Current Opinion in Microbiology, vol. 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
In: The ISME Journal, vol. 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=1.16âMb), with an unusually low genomic G+C mol% (âŒ42%). 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 (âŒ15%) 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=1.16âMb), with an unusually low genomic G+C mol% (âŒ42%). 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 (âŒ15%) 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
In: PLoS ONE, vol. 7, no. 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
In: Scientific Report, vol. 1, no. 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 784â
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 784â
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
In: PLoS ONE, vol. 6, no. 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
In: The ISME Journal, vol. 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 predatorâprey 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
In: PLOS Genetics, vol. 10, no. 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
In: Science, vol. 333, no. 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 oceanâs 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 oceanâs 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
In: PLoS ONE, vol. 6, no. 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
In: The ISME Journal, vol. 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 10â23% 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
In: Science, vol. 332, no. 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
In: PLoS ONE, vol. 6, no. 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 (â€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 (â€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
In: Science, vol. 331, no. 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
In: The ISME Journal, vol. 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
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
In: Molecular biology and evolution, vol. 27, no. 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 prokaryoteâeukaryote (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 SynechococcusâProchlorococcus 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.
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 prokaryoteâeukaryote (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 SynechococcusâProchlorococcus 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
In: PLoS ONE, vol. 4, no. 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
In: Current Opinion in Microbiology, vol. 11, no. 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
In: PNAS, vol. 104, no. 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 88â97% 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.