Stepanauskas Ramunas

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  • Article
    Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system
    (Frontiers Media, 2019-06-12) Labonté, Jessica M. ; Pachiadaki, Maria G. ; Fergusson, Elizabeth ; McNichol, Jesse C. ; Grosche, Ashley ; Gulmann, Lara K. ; Vetriani, Costantino ; Sievert, Stefan M. ; Stepanauskas, Ramunas
    Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.
  • Article
    Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres
    (Public Library of Science, 2014-04-17) Swan, Brandon K. ; Chaffin, Mark D. ; Martinez-Garcia, Manuel ; Morrison, Hilary G. ; Field, Erin K. ; Poulton, Nicole J. ; Masland, E. Dashiell P. ; Harris, Christopher C. ; Sczyrba, Alexander ; Chain, Patrick S. G. ; Koren, Sergey ; Woyke, Tanja ; Stepanauskas, Ramunas
    Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.
  • Article
    Ancestral absence of electron transport chains in Patescibacteria and DPANN
    (Frontiers Media, 2020-08-17) Beam, Jacob P. ; Becraft, Eric D. ; Brown, Julia M. ; Schulz, Frederik ; Jarett, Jessica K. ; Bezuidt, Oliver ; Poulton, Nicole J. ; Clark, Kayla ; Dunfield, Peter F. ; Ravin, Nikolai V. ; Spear, John R. ; Hedlund, Brian P. ; Kormas, Konstantinos Ar. ; Sievert, Stefan M. ; Elshahed, Mostafa S. ; Barton, Hazel A. ; Stott, Matthew B. ; Eisen, Jonathan A. ; Moser, Duane P. ; Onstott, Tullis C. ; Woyke, Tanja ; Stepanauskas, Ramunas
    Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell–cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.
  • Dataset
    Single amplified genomes (SAGs) of chemoautotrophs from global deep sea samples (Dark ocean chemoautotrophs project)
    (Biological and Chemical Oceanography Data Management Office (BCO-DMO). Contact:, 2021-04-01) Stepanauskas, Ramunas
    This dataset includes accession numbers archived at IMG/M (Integrated Microbial Genomes and Microbiome Samples) at the US Dept. of Energy's Joint Genome Institute. Additional information includes the ocean depth, latitude and longitude, the assembled genome size and count, and the scaffold count. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at:
  • Article
    Insights into the phylogeny and coding potential of microbial dark matter
    (Nature Publishing Group, 2013-07-14) Rinke, Christian ; Schwientek, Patrick ; Sczyrba, Alexander ; Ivanova, Natalia N. ; Anderson, Iain J. ; Cheng, Jan-Fang ; Darling, Aaron ; Malfatti, Stephanie A. ; Swan, Brandon K. ; Gies, Esther A. ; Dodsworth, Jeremy A. ; Hedlund, Brian P. ; Tsiamis, Georgios ; Sievert, Stefan M. ; Liu, Wen-Tso ; Eisen, Jonathan A. ; Hallam, Steven J. ; Kyrpides, Nikos C. ; Stepanauskas, Ramunas ; Rubin, Edward M. ; Hugenholtz, Philip ; Woyke, Tanja
    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.
  • Article
    Hiding in plain sight: the globally distributed bacterial candidate phylum PAUC34f
    (Frontiers Media, 2020-03-12) Chen, Michael L. ; Becraft, Eric D. ; Pachiadaki, Maria G. ; Brown, Julia M. ; Jarett, Jessica K. ; Gasol, Josep M. ; Ravin, Nikolai V. ; Moser, Duane P. ; Nunoura, Takuro ; Herndl, Gerhard J. ; Woyke, Tanja ; Stepanauskas, Ramunas
    Bacterial candidate phylum PAUC34f was originally discovered in marine sponges and is widely considered to be composed of sponge symbionts. Here, we report 21 single amplified genomes (SAGs) of PAUC34f from a variety of environments, including the dark ocean, lake sediments, and a terrestrial aquifer. The diverse origins of the SAGs and the results of metagenome fragment recruitment suggest that some PAUC34f lineages represent relatively abundant, free-living cells in environments other than sponge microbiomes, including the deep ocean. Both phylogenetic and biogeographic patterns, as well as genome content analyses suggest that PAUC34f associations with hosts evolved independently multiple times, while free-living lineages of PAUC34f are distinct and relatively abundant in a wide range of environments.
  • Article
    Phosphonate production by marine microbes: exploring new sources and potential function
    (National Academy of Sciences, 2022-03-07) Acker, Marianne ; Hogle, Shane L. ; Berube, Paul M. ; Hackl, Thomas ; Coe, Allison ; Stepanauskas, Ramunas ; Chisholm, Sallie W. ; Repeta, Daniel J.
    Phosphonates are organophosphorus metabolites with a characteristic C-P bond. They are ubiquitous in the marine environment, their degradation broadly supports ecosystem productivity, and they are key components of the marine phosphorus (P) cycle. However, the microbial producers that sustain the large oceanic inventory of phosphonates as well as the physiological and ecological roles of phosphonates are enigmatic. Here, we show that phosphonate synthesis genes are rare but widely distributed among diverse bacteria and archaea, including Prochlorococcus and SAR11, the two major groups of bacteria in the ocean. In addition, we show that Prochlorococcus can allocate over 40% of its total cellular P-quota toward phosphonate production. However, we find no evidence that Prochlorococcus uses phosphonates for surplus P storage, and nearly all producer genomes lack the genes necessary to degrade and assimilate phosphonates. Instead, we postulate that phosphonates are associated with cell-surface glycoproteins, suggesting that phosphonates mediate ecological interactions between the cell and its surrounding environment. Our findings indicate that the oligotrophic surface ocean phosphonate pool is sustained by a relatively small fraction of the bacterioplankton cells allocating a significant portion of their P quotas toward secondary metabolism and away from growth and reproduction.
  • Article
    Oceanic crustal fluid single cell genomics complements metagenomic and metatranscriptomic surveys with orders of magnitude less sample volume
    (Frontiers Media, 2022-01-24) D'Angelo, Timothy ; Goordial, Jacqueline M. ; Poulton, Nicole J. ; Seyler, Lauren M. ; Huber, Julie A. ; Stepanauskas, Ramunas ; Orcutt, Beth N.
    Fluids circulating through oceanic crust play important roles in global biogeochemical cycling mediated by their microbial inhabitants, but studying these sites is challenged by sampling logistics and low biomass. Borehole observatories installed at the North Pond study site on the western flank of the Mid-Atlantic Ridge have enabled investigation of the microbial biosphere in cold, oxygenated basaltic oceanic crust. Here we test a methodology that applies redox-sensitive fluorescent molecules for flow cytometric sorting of cells for single cell genomic sequencing from small volumes of low biomass (approximately 103 cells ml–1) crustal fluid. We compare the resulting genomic data to a recently published paired metagenomic and metatranscriptomic analysis from the same site. Even with low coverage genome sequencing, sorting cells from less than one milliliter of crustal fluid results in similar interpretation of dominant taxa and functional profiles as compared to ‘omics analysis that typically filter orders of magnitude more fluid volume. The diverse community dominated by Gammaproteobacteria, Bacteroidetes, Desulfobacterota, Alphaproteobacteria, and Zetaproteobacteria, had evidence of autotrophy and heterotrophy, a variety of nitrogen and sulfur cycling metabolisms, and motility. Together, results indicate fluorescence activated cell sorting methodology is a powerful addition to the toolbox for the study of low biomass systems or at sites where only small sample volumes are available for analysis.
  • Dataset
    Single amplified genomes (SAGs) of microbial cells isolated from deep-sea hydrothermal vent 'Crab Spa', East Pacific Rise, Pacific Ocean from R/V Atlantis AT15-38 and AT26-10, 2008 and 2014 (Microbial Communities at Deep-Sea Vents project)
    (Biological and Chemical Oceanography Data Management Office (BCO-DMO). Contact:, 2021-04-07) Stepanauskas, Ramunas
    Single amplified genomes (SAGs) of microbial cells isolated from deep-sea hydrothermal vent 'Crab Spa', East Pacific Rise, Pacific Ocean from R/V Atlantis AT15-38 and AT26-10, 2008 and 2014 (Microbial Communities at Deep-Sea Vents project) For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at:
  • Article
    Pangenomics analysis reveals diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria
    (American Society for Microbiology, 2020-01-07) Saw, Jimmy H. W. ; Nunoura, Takuro ; Hirai, Miho ; Takaki, Yoshihiro ; Parsons, Rachel ; Michelsen, Michelle ; Longnecker, Krista ; Kujawinski, Elizabeth B. ; Stepanauskas, Ramunas ; Landry, Zachary ; Carlson, Craig A. ; Giovannoni, Stephen J.
    It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns. IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Information about microbial interactions with this vast resource is limited, despite broad recognition that DOM turnover has a major impact on the global carbon cycle. To explain patterns in the genomes of marine bacteria, we propose hypothetical metabolic pathways for the oxidation of organic molecules that are resistant to oxidation via common pathways. The hypothetical schemes we propose suggest new metabolic pathways and classes of compounds that could be important for understanding the distribution of organic carbon throughout the biosphere. These genome-based schemes will remain hypothetical until evidence from experimental cell biology can be gathered to test them. Our findings also fundamentally change our understanding of the ecology of SAR202 bacteria, showing that metabolically diverse variants of these cells occupy niches spanning all depths and are not relegated to the dark ocean.