Publications

@article{C2013,

title = {Insights into the phylogeny and coding potential of microbial dark matter},

author = {Rinke C and Schwientek P and Sczyrba A and Ivanova NN and Anderson IJ and Cheng J-F and Darling A and Malfatti S and Swan BK and Gies EA and Dodsworth JA and Hedlund BP and Tsiamis G and Sievert SM and Liu W-T and Eisen JA and Hallam S and Kyrpides N and Stepanauskas R and Rubin E and Hugenholtz P and Woyke T},

url = {https://www.nature.com/articles/nature12352},

doi = {10.1038/nature12352},

year  = {2013},

date = {2013-07-14},

journal = {Nature},

volume = {499},

pages = {431-437},

abstract = {Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@article{KG2013,

title = {Predominant archaea in marine sediments degrade detrital proteins},

author = {Lloyd KG and Schreiber L and Petersen DG and Kjeldsen K and Lever MA and Stepanauskas R and Richter M and Kleindienst S and Lenk S and Schramm A and Jorgensen BB},

url = {https://www.nature.com/articles/nature12033},

doi = {10.1038/nature12033},

year  = {2013},

date = {2013-04-11},

journal = {Nature},

volume = {496},

pages = {215-182},

abstract = {Half of the microbial cells in the Earth’s oceans are found in sediments1. Many of these cells are members of the Archaea2, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota3 and Aigarchaeota4, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@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}

}

@article{LG2012,

title = {Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton},

author = {Garcia LG and McMahon KD and Martinez-Garcia M and Srivastava A and Sczyrba A and Stepanauskas R and Grossart HP and Woyke T and Warnecke F},

url = {https://www.nature.com/articles/ismej201286},

doi = {10.1038/ismej.2012.86},

year  = {2012},

date = {2012-07-19},

journal = {The ISME Journal},

volume = {7},

pages = {137-147},

abstract = {Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@article{R2011,

title = {New abundant microbial groups in aquatic hypersaline environments},

author = {Ghai R and Pasic L and Fernandez AB and Martin-Cuadrado AB and Mizuno CM and McMahon KD and Papke RT and Stepanauskas R and Rodriguez-Brito B and Rohwer F and Sanchez-Porro C and Ventosa A and Rodriguez-Valera F},

url = {https://www.nature.com/articles/srep00135},

doi = {10.1038/srep00135},

year  = {2011},

date = {2011-10-31},

journal = {Scientific Report},

volume = {1},

number = {135},

abstract = {We describe the microbiota of two hypersaline saltern ponds, one of intermediate salinity (19%) and a NaCl saturated crystallizer pond (37%) using pyrosequencing. The analyses of these metagenomes (nearly 784 Mb) reaffirmed the vast dominance of Haloquadratum walsbyi but also revealed novel, abundant and previously unsuspected microbial groups. We describe for the first time, a group of low GC Actinobacteria, related to freshwater Actinobacteria, abundant in low and intermediate salinities. Metagenomic assembly revealed three new abundant microbes: a low-GC euryarchaeon with the lowest GC content described for any euryarchaeon, a high-GC euryarchaeon and a gammaproteobacterium related to Alkalilimnicola and Nitrococcus. Multiple displacement amplification and sequencing of the genome from a single archaeal cell of the new low GC euryarchaeon suggest a photoheterotrophic and polysaccharide-degrading lifestyle and its relatedness to the recently described lineage of Nanohaloarchaea. These discoveries reveal the combined power of an unbiased metagenomic and single cell genomic approach.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@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}

}

@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}

}

@article{BK2011,

title = {Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean},

author = {Swan BK and Martinez-Garcia M and Preston CM and Sczyrba A and Woyke T and Lamy D and Reinthaler T and Poulton NJ and Masland D and Lluesma Gomez M and Sieracki ME and DeLong EF and Herndl GJ and Stepanauskas R},

url = {http://science.sciencemag.org/content/333/6047/1296},

doi = {10.1126/science.1203690},

year  = {2011},

date = {2011-09-02},

journal = {Science},

volume = {333},

number = {6047},

pages = {1296-1300},

abstract = {Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C1 metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean’s largest habitat.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@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}

}

@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}

}

@article{EJ2011,

title = {What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH},

author = {Fleming EJ and Langdon AE and Martinez-Garcia M and Stepanauskas R and Poulton N and Masland D and Emerson D},

url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017769},

doi = {https://doi.org/10.1371/journal.pone.0017769},

year  = {2011},

date = {2011-03-17},

journal = {PLoS ONE},

volume = {6},

number = {3},

pages = {e17769},

abstract = {Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}

@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}

}