Publications

@article{T2024,

title = {Genomic representativeness and chimerism in large collections of SAGs and MAGs of marine prokaryoplankton},

author = {Chang T and Gavelis GS and Brown JM and Stepanauskas R },

url = {https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-024-01848-3},

doi = {https://doi.org/10.1186/s40168-024-01848-3},

issn = {2049-2618},

year  = {2024},

date = {2024-07-15},

urldate = {2024-07-15},

journal = {Microbiome},

volume = {12},

number = {126},

abstract = {Background

Single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) are the predominant sources of information about the coding potential of uncultured microbial lineages, but their strengths and limitations remain poorly understood. Here, we performed a direct comparison of two previously published collections of thousands of SAGs and MAGs obtained from the same, global environment.



Results

We found that SAGs were less prone to chimerism and more accurately reflected the relative abundance and the pangenome content of microbial lineages inhabiting the epipelagic of the tropical and subtropical ocean, as compared to MAGs. SAGs were also better suited to link genome information with taxa discovered through 16S rRNA amplicon analyses. Meanwhile, MAGs had the advantage of more readily recovering genomes of rare lineages.



Conclusions

Our analyses revealed the relative strengths and weaknesses of the two most commonly used genome recovery approaches in environmental microbiology. These considerations, as well as the need for better tools for genome quality assessment, should be taken into account when designing studies and interpreting data that involve SAGs or MAGs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{MG2024,

title = {Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe},

author = {Miyada MG and Choi Y and Stepanauskas R and Woyke T and La Clair JJ and Burkart MD},

url = {https://pubs.acs.org/doi/10.1021/acschembio.4c00169},

doi = {10.1021/acschembio.4c00169},

year  = {2024},

date = {2024-06-23},

journal = {ACS Chemical Biology},

volume = {18},

issue = {7},

abstract = {Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP\textendashenzyme interactions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{MR2024,

title = {Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem},

author = {Lindsay MR and D\'Angelo T and Munson-McGee JH and Saidi-Mehrabad A and Devlin M and McGonigle J and Goodell E and Herring M and Lubelczyk LC and Mascena C and Brown JM and Gavelis G and Liu J and Yousavich DJ and Hamilton-Brehm SD and Hedlund BP and Lang S and Treude T and Poulton NJ and Stepanauskas R and Moser DP and Emerson D and Orcutt BN},

url = {https://www.pnas.org/doi/10.1073/pnas.2309636121},

doi = {https://doi.org/10.1073/pnas.2309636121},

year  = {2024},

date = {2024-04-04},

journal = {PNAS},

volume = {121},

number = {15},

pages = {e2309636121},

abstract = {Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~103 cells mL−1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell−1 h−1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L−1 d−1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{A2023,

title = {Interplay between autotrophic and heterotrophic prokaryotic metabolism in the bathypelagic realm revealed by metatranscriptomic analyses},

author = {Srivastava A and De Corte D and Garcia JAL and Swan BK and Stepanauskas R and Herndl GJ and Sintes E },

url = {https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-023-01688-7},

doi = {https://doi.org/10.1186/s40168-023-01688-7},

issn = {2049-2618},

year  = {2023},

date = {2023-11-04},

journal = {Microbiome},

volume = {11},

number = {239},

abstract = {Background

Heterotrophic microbes inhabiting the dark ocean largely depend on the settling of organic matter from the sunlit ocean. However, this sinking of organic materials is insufficient to cover their demand for energy and alternative sources such as chemoautotrophy have been proposed. Reduced sulfur compounds, such as thiosulfate, are a potential energy source for both auto- and heterotrophic marine prokaryotes.



Methods

Seawater samples were collected from Labrador Sea Water (LSW, ~ 2000 m depth) in the North Atlantic and incubated in the dark at in situ temperature unamended, amended with 1 µM thiosulfate, or with 1 µM thiosulfate plus 10 µM glucose and 10 µM acetate (thiosulfate plus dissolved organic matter, DOM). Inorganic carbon fixation was measured in the different treatments and samples for metatranscriptomic analyses were collected after 1 h and 72 h of incubation.



Results

Amendment of LSW with thiosulfate and thiosulfate plus DOM enhanced prokaryotic inorganic carbon fixation. The energy generated via chemoautotrophy and heterotrophy in the amended prokaryotic communities was used for the biosynthesis of glycogen and phospholipids as storage molecules. The addition of thiosulfate stimulated unclassified bacteria, sulfur-oxidizing Deltaproteobacteria (SAR324 cluster bacteria), Epsilonproteobacteria (Sulfurimonas sp.), and Gammaproteobacteria (SUP05 cluster bacteria), whereas, the amendment with thiosulfate plus DOM stimulated typically copiotrophic Gammaproteobacteria (closely related to Vibrio sp. and Pseudoalteromonas sp.).



Conclusions

The gene expression pattern of thiosulfate utilizing microbes specifically of genes involved in energy production via sulfur oxidation and coupled to CO2 fixation pathways coincided with the change in the transcriptional profile of the heterotrophic prokaryotic community (genes involved in promoting energy storage), suggesting a fine-tuned metabolic interplay between chemoautotrophic and heterotrophic microbes in the dark ocean.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JM2023,

title = {Flotillin-associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes},

author = {Haro-Moreno JM and L\'{o}pez-P\'{e}rez M and Alekseev A and Podoliak E and Kovalev K and Gordeliy V and Stepanauskas R and Rodriguez-Valera F},

url = {https://pubmed.ncbi.nlm.nih.gov/37222519/},

doi = {10.1128/msystems.00008-23},

year  = {2023},

date = {2023-06-29},

journal = {mSystems},

volume = {8},

number = {e0000823},

issue = {3},

abstract = {Microbial rhodopsins are found more than once in a single genome (paralogs) often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59, and the Gammaproteobacteria Pseudothioglobus SAGs. These genomes always had a bona fide proteorhodopsin and a separate cluster of genes containing a second rhodopsin associated with a predicted flotillin coding gene and have thus been named flotillin-associated rhodopsins (FArhodopsins). Although they are members of the proteorhodopsin protein family, they form a separate clade within that family and are quite divergent from known proton-pumping proteorhodopsins. They contain either DTT, DTL, or DNI motifs in their key functional amino acids. FArhodopsins are mainly associated with the lower layers of the epipelagic zone. All marine FArhodopsins had the retinal binding lysine, but we found relatives in freshwater metagenomes lacking this key amino acid. AlphaFold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less. Freshwater FArhodopsins were more diverse than marine ones, but we could not determine if there were other rhodopsins in the genome due to the lack of SAGs or isolates. Although the function of FArhodopsins could not be established, their conserved genomic context indicated involvement in the formation of membrane microdomains. The conservation of FArhodopsins in diverse and globally abundant microorganisms suggests that they may be important in the adaptation to the twilight zone of aquatic environments. IMPORTANCE Rhodopsins have been shown to play a key role in the ecology of aquatic microbes. Here, we describe a group of widespread rhodopsins in aquatic microbes associated with dim light conditions. Their characteristic genomic context found in both marine and freshwater environments indicates a novel potential involvement in membrane microstructure that could be important for the function of the coexisting proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to a drastically different physiological role.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{AE2023,

title = {Life strategies for Aminicenantia in subseafloor oceanic crust},

author = {Booker AE and D’Angelo T and Adams-Beyea A and Brown JM and Nigro O and Rapp\'{e} MS and Stepanauskas R and Orcutt BN},

url = {https://www.nature.com/articles/s41396-023-01454-5},

doi = {https://doi.org/10.1038/s41396-023-01454-5},

issn = {1751-7370 (online)},

year  = {2023},

date = {2023-06-16},

journal = {The ISME Journal},

volume = {17},

pages = {1406-1415},

abstract = {After decades studying the microbial “deep biosphere” in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use “primordial” siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{M2023,

title = {Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium},

author = {D\v{z}unkov\'{a} M and La Clair JJ and Tyml T and Doud D and Schulz F and Piquer-Esteban S and Porcel Sanchis D and Osborn A and Robinson D and Louie KB and Bowen BP and Bowers RM and Lee J and Arnau V and D\'{i}az-Villanueva W and Stepanauskas R and Gosliner T and Date SV and Northen TR and Cheng JF and Burkart MD and Woyke T },

url = {https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-023-01560-8},

doi = {https://doi.org/10.1186/s40168-023-01560-8},

issn = {2049-2618},

year  = {2023},

date = {2023-06-13},

journal = {Microbiome},

volume = {11},

number = {130},

abstract = {Nudibranchs comprise a group of \> 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{J2023,

title = {A compendium of bacterial and archaeal single-cell amplified genomes from oxygen deficient marine waters},

author = {Anstett J and Plominsky AM and DeLong EF and Kiesser A and J\"{u}rgens K and Morgan-Lang C and Stepanauskas R and Stewart FJ and Ulloa O and Woyke T and Malmstrom R and Hallam SJ },

url = {https://www.nature.com/articles/s41597-023-02222-y},

doi = {https://doi.org/10.1038/s41597-023-02222-y},

issn = {2052-4463 (online)},

year  = {2023},

date = {2023-05-27},

journal = {Scientific Data},

volume = {10},

number = {332},

abstract = {Oxygen-deficient marine waters referred to as oxygen minimum zones (OMZs) or anoxic marine zones (AMZs) are common oceanographic features. They host both cosmopolitan and endemic microorganisms adapted to low oxygen conditions. Microbial metabolic interactions within OMZs and AMZs drive coupled biogeochemical cycles resulting in nitrogen loss and climate active trace gas production and consumption. Global warming is causing oxygen-deficient waters to expand and intensify. Therefore, studies focused on microbial communities inhabiting oxygen-deficient regions are necessary to both monitor and model the impacts of climate change on marine ecosystem functions and services. Here we present a compendium of 5,129 single-cell amplified genomes (SAGs) from marine environments encompassing representative OMZ and AMZ geochemical profiles. Of these, 3,570 SAGs have been sequenced to different levels of completion, providing a strain-resolved perspective on the genomic content and potential metabolic interactions within OMZ and AMZ microbiomes. Hierarchical clustering confirmed that samples from similar oxygen concentrations and geographic regions also had analogous taxonomic compositions, providing a coherent framework for comparative community analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{T2023b,

title = {Replicated life-history patterns and subsurface origins of the bacterial sister phyla Nitrospirota and Nitrospinota},

author = {D\'Angelo T and Goordial J and Lindsay MR and McGonigle J and Booker A and Moser D and Stepanauskas R and Orcutt BN},

url = {https://www.nature.com/articles/s41396-023-01397-x},

doi = {https://doi.org/10.1038/s41396-023-01397-x},

issn = {1751-7370 (online)},

year  = {2023},

date = {2023-04-03},

journal = {The ISME Journal},

volume = {17},

pages = {891-902},

abstract = {The phyla Nitrospirota and Nitrospinota have received significant research attention due to their unique nitrogen metabolisms important to biogeochemical and industrial processes. These phyla are common inhabitants of marine and terrestrial subsurface environments and contain members capable of diverse physiologies in addition to nitrite oxidation and complete ammonia oxidation. Here, we use phylogenomics and gene-based analysis with ancestral state reconstruction and gene-tree\textendashspecies-tree reconciliation methods to investigate the life histories of these two phyla. We find that basal clades of both phyla primarily inhabit marine and terrestrial subsurface environments. The genomes of basal clades in both phyla appear smaller and more densely coded than the later-branching clades. The extant basal clades of both phyla share many traits inferred to be present in their respective common ancestors, including hydrogen, one-carbon, and sulfur-based metabolisms. Later-branching groups, namely the more frequently studied classes Nitrospiria and Nitrospinia, are both characterized by genome expansions driven by either de novo origination or laterally transferred genes that encode functions expanding their metabolic repertoire. These expansions include gene clusters that perform the unique nitrogen metabolisms that both phyla are most well known for. Our analyses support replicated evolutionary histories of these two bacterial phyla, with modern subsurface environments representing a genomic repository for the coding potential of ancestral metabolic traits.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{CO2023,

title = {Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota},

author = {Seymour CO and Palmer M and Becraft ED and Stepanauskas R and Friel AD and Schulz F and Woyke T and Eloe-Fadrosh E and Lai D and Jiao J-Y and Hua Z-S and Liu L and Lian Z-H and Li W-J and Chuvochina M and Finley BK and Koch BJ and Schwartz E and Dijkstra P and Moser DP and Hungate BA and Hedlund BP },

url = {https://www.nature.com/articles/s41564-022-01319-1},

doi = {https://doi.org/10.1038/s41564-022-01319-1},

year  = {2023},

date = {2023-03-16},

journal = {Nature Microbiology},

abstract = {Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 μm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{T2023,

title = {Novel integrative elements and genomic plasticity in ocean ecosystems},

author = {Hackl T and Laurenceau R and Ankenbrand MJ and Bliem C and Cariani Z and Thomas E and Dooley KD and Arellano AA and Hogle SL and Berube P and Leventhal GE and Luo E and Eppley JM and Zayed AA and Beaulaurier J and Stepanauskas R and Sullivan MB and DeLong EF and Biller SJ and Chisholm SW},

url = {https://www.sciencedirect.com/science/article/abs/pii/S0092867422015197?via%3Dihub},

doi = {https://doi.org/10.1016/j.cell.2022.12.006},

issn = {0092-8674},

year  = {2023},

date = {2023-01-05},

journal = {Cell},

volume = {186},

issue = {1},

pages = {47-62.e16},

abstract = {Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons\textemdash“tycheposons”\textemdashsome of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands\textemdashkey reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Munson-McGee2022,

title = {Decoupling of respiration rates and abundance in marine prokaryoplankton},

author = {Munson-McGee J.H. and Lindsay M.R. and Sintes E and Brown J.M. and D'Angelo T and Brown J and Lubelczyk L.C. and Tomko P and Emerson D and Orcutt B.N. and Poulton N.J. and Herndl G.J. and Stepanauskas R},

url = {https://www.nature.com/articles/s41586-022-05505-3#citeas},

doi = {https://doi.org/10.1038/s41586-022-05505-3},

year  = {2022},

date = {2022-12-07},

urldate = {2022-12-07},

journal = {Nature},

volume = {612},

pages = {764-770},

abstract = {The ocean\textendashatmosphere exchange of CO2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton)1,2,3, their respiration usually is measured in bulk and treated as a ‘black box’ in global biogeochemical models4; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO2 for its energy demands and growth may be lower than commonly assumed and variable among lineages.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{BJ2022,

title = {Thousands of small, novel genes predicted in global phage genomes},

author = {Fremin BJ and Bhatt AS and Kyrpides NC and Global Phage Small Open Reading Frame C },

url = {https://www.sciencedirect.com/science/article/pii/S2211124722007707?via%3Dihub},

doi = {https://doi.org/10.1016/j.celrep.2022.110984},

year  = {2022},

date = {2022-06-21},

journal = {Cell Reports},

volume = {39},

number = {110984},

issue = {12},

abstract = {Small genes (\<150 nucleotides) have been systematically overlooked in phage genomes. We employ a large-scale comparative genomics approach to predict \>40,000 small-gene families in ∼2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{S2022,

title = {Single cell genome sequencing of laboratory mouse microbiota improves taxonomic and functional resolution of this model microbial community},

author = {Lyalina S and Stepanauskas R and Wu F and Sanjabi S and Pollard KS },

url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261795},

doi = {https://doi.org/10.1371/journal.pone.0261795},

year  = {2022},

date = {2022-04-13},

journal = {PLoS One },

volume = {17},

number = {e0261795},

issue = {4},

abstract = {Laboratory mice are widely studied as models of mammalian biology, including the microbiota. However, much of the taxonomic and functional diversity of the mouse gut microbiome is missed in current metagenomic studies, because genome databases have not achieved a balanced representation of the diverse members of this ecosystem. Towards solving this problem, we used flow cytometry and low-coverage sequencing to capture the genomes of 764 single cells from the stool of three laboratory mice. From these, we generated 298 high-coverage microbial genome assemblies, which we annotated for open reading frames and phylogenetic placement. These genomes increase the gene catalog and phylogenetic breadth of the mouse microbiota, adding 135 novel species with the greatest increase in diversity to the Muribaculaceae and Bacteroidaceae families. This new diversity also improves the read mapping rate, taxonomic classifier performance, and gene detection rate of mouse stool metagenomes. The novel microbial functions revealed through our single-cell genomes highlight previously invisible pathways that may be important for life in the murine gastrointestinal tract.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Magnuson2022,

title = {Active lithoautotrophic and methane-oxidizing microbial community in an anoxic, sub-zero, and hypersaline High Arctic spring},

editor = {Magnuson, E and Altshuler, I and Fern\'{a}ndez-Mart\'{i}nez, M.\'{A}. and Chen, Y and Maggiori, C and Goordial, J and Whyte, L.G. },

doi = {https://doi.org/10.1038/s41396-022-01233-8},

year  = {2022},

date = {2022-04-08},

journal = {ISME Journal},

volume = {16},

pages = {1798-1808},

abstract = {Lost Hammer Spring, located in the High Arctic of Nunavut, Canada, is one of the coldest and saltiest terrestrial springs discovered to date. It perennially discharges anoxic (\<1 ppm dissolved oxygen), sub-zero (~−5 °C), and hypersaline (~24% salinity) brines from the subsurface through up to 600 m of permafrost. The sediment is sulfate-rich (1 M) and continually emits gases composed primarily of methane (~50%), making Lost Hammer the coldest known terrestrial methane seep and an analog to extraterrestrial habits on Mars, Europa, and Enceladus. A multi-omics approach utilizing metagenome, metatranscriptome, and single-amplified genome sequencing revealed a rare surface terrestrial habitat supporting a predominantly lithoautotrophic active microbial community driven in part by sulfide-oxidizing Gammaproteobacteria scavenging trace oxygen. Genomes from active anaerobic methane-oxidizing archaea (ANME-1) showed evidence of putative metabolic flexibility and hypersaline and cold adaptations. Evidence of anaerobic heterotrophic and fermentative lifestyles were found in candidate phyla DPANN archaea and CG03 bacteria genomes. Our results demonstrate Mars-relevant metabolisms including sulfide oxidation, sulfate reduction, anaerobic oxidation of methane, and oxidation of trace gases (H2, CO2) detected under anoxic, hypersaline, and sub-zero ambient conditions, providing evidence that similar extant microbial life could potentially survive in similar habitats on Mars.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{M2022,

title = {Phosphonate production by marine microbes: Exploring new sources and potential function},

author = {Acker M and Hogle SL and Berube PM and Hackl T and Coe A and Stepanauskas R and Chisholm SW and Repeta DJ},

url = {https://www.pnas.org/doi/10.1073/pnas.2113386119},

doi = {https://doi.org/10.1073/pnas.2113386119},

year  = {2022},

date = {2022-03-07},

urldate = {2022-03-07},

journal = {PNAS},

volume = {119},

number = {11},

pages = {e2113386119},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{T2022,

title = {Oceanic crustal fluid single cell genomics complements metagenomic and metatranscriptomic surveys with orders of magnitude less sample volume},

author = {D’Angelo T and Goordial J and Poulton N and Seyler L and Huber JA and Stepanauskas R and Orcutt BN},

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2021.738231/full},

doi = {https://doi.org/10.3389/fmicb.2021.738231},

year  = {2022},

date = {2022-01-24},

journal = {Frontiers in Microbiology},

volume = {12},

number = {738231},

abstract = {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\textendash1) 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{C2022b,

title = {Phylogenetically and functionally diverse microorganisms reside under the Ross Ice Shelf},

author = {Mart\'{i}nez-P\'{e}rez C and Greening C and Bay SK and Lappan RJ and Zhao Z and De Corte D and Hulbe C and Ohneiser C and Stevens C and Thomson B and Stepanauskas R and Gonz\'{a}lez JM and Logares R and Herndl GJ and Morales SE and Baltar F},

url = {https://www.nature.com/articles/s41467-021-27769-5},

doi = {https://doi.org/10.1038/s41467-021-27769-5},

year  = {2022},

date = {2022-01-10},

journal = {Nature Communications},

volume = {13},

number = {117},

abstract = {Throughout coastal Antarctica, ice shelves separate oceanic waters from sunlight by hundreds of meters of ice. Historical studies have detected activity of nitrifying microorganisms in oceanic cavities below permanent ice shelves. However, little is known about the microbial composition and pathways that mediate these activities. In this study, we profiled the microbial communities beneath the Ross Ice Shelf using a multi-omics approach. Overall, beneath-shelf microorganisms are of comparable abundance and diversity, though distinct composition, relative to those in the open meso- and bathypelagic ocean. Production of new organic carbon is likely driven by aerobic lithoautotrophic archaea and bacteria that can use ammonium, nitrite, and sulfur compounds as electron donors. Also enriched were aerobic organoheterotrophic bacteria capable of degrading complex organic carbon substrates, likely derived from in situ fixed carbon and potentially refractory organic matter laterally advected by the below-shelf waters. Altogether, these findings uncover a taxonomically distinct microbial community potentially adapted to a highly oligotrophic marine environment and suggest that ocean cavity waters are primarily chemosynthetically-driven systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{C2022,

title = {Phylogenetically and functionally diverse microorganisms reside under the Ross Ice Shelf},

author = {Mart\'{i}nez-P\'{e}rez C and Greening C and Bay SK and Lappan RJ and Zhao Z and De Corte D and Hulbe C and Ohneiser C and Stevens C and Thomson B and Stepanauskas R and Gonz\'{a}lez JM and Logares R and Herndl GJ and Morales SE and Baltar F},

url = {https://www.nature.com/articles/s41467-021-27769-5},

doi = {https://doi.org/10.1038/s41467-021-27769-5},

year  = {2022},

date = {2022-01-10},

urldate = {2022-01-10},

journal = {Nature Communications},

volume = {13},

number = {117},

abstract = {Throughout coastal Antarctica, ice shelves separate oceanic waters from sunlight by hundreds of meters of ice. Historical studies have detected activity of nitrifying microorganisms in oceanic cavities below permanent ice shelves. However, little is known about the microbial composition and pathways that mediate these activities. In this study, we profiled the microbial communities beneath the Ross Ice Shelf using a multi-omics approach. Overall, beneath-shelf microorganisms are of comparable abundance and diversity, though distinct composition, relative to those in the open meso- and bathypelagic ocean. Production of new organic carbon is likely driven by aerobic lithoautotrophic archaea and bacteria that can use ammonium, nitrite, and sulfur compounds as electron donors. Also enriched were aerobic organoheterotrophic bacteria capable of degrading complex organic carbon substrates, likely derived from in situ fixed carbon and potentially refractory organic matter laterally advected by the below-shelf waters. Altogether, these findings uncover a taxonomically distinct microbial community potentially adapted to a highly oligotrophic marine environment and suggest that ocean cavity waters are primarily chemosynthetically-driven systems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RM2021b,

title = {Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution},

author = {Bowers RM and Nayfach S and Schulz F and Jungbluth SP and Ruhl IA and Sheremet A and Lee J and Goudeau D and Eloe-Fadrosh EA and Stepanauskas R and Malmstrom RR and Kyrpides NC and Dunfield PF and Woyke T},

url = {https://www.nature.com/articles/s41396-021-01178-4},

doi = {https://doi.org/10.1038/s41396-021-01178-4},

year  = {2021},

date = {2021-12-30},

journal = {The ISME Journal},

volume = {16},

pages = {1337-1347},

abstract = {With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek’s Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RM2021,

title = {Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell resolution},

author = {Bowers RM and Nayfach S and Schulz F and Jungbluth SP and Ruhl IA and Lee J and Goudeau D and Eloe-Fadrosh EA and Stepanauskas R and Malmstrom RR and Krypides NC and Dunfield PF and Woyke T},

url = {https://www.nature.com/articles/s41396-021-01178-4},

doi = {https://doi.org/10.1038/s41396-021-01178-4},

year  = {2021},

date = {2021-12-30},

journal = {ISME Journal},

abstract = {With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek’s Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{R2021,

title = {Clean room microbiome complexity impacts planetary protection bioburden},

author = {Hendrickson R and Urbaniak C and Minich JJ and Aronson HS and Martino C and Stepanauskas R and Knight R and Venkateswaran K},

url = {https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-021-01159-x},

doi = {https://doi.org/10.1186/s40168-021-01159-x},

isbn = {2049-2618},

year  = {2021},

date = {2021-12-04},

urldate = {2021-12-04},

journal = {Microbiome},

volume = {9},

number = {238},

abstract = {Background

The Spacecraft Assembly Facility (SAF) at the NASA’s Jet Propulsion Laboratory is the primary cleanroom facility used in the construction of some of the planetary protection (PP)-sensitive missions developed by NASA, including the Mars 2020 Perseverance Rover that launched in July 2020. SAF floor samples (n=98) were collected, over a 6-month period in 2016 prior to the construction of the Mars rover subsystems, to better understand the temporal and spatial distribution of bacterial populations (total, viable, cultivable, and spore) in this unique cleanroom.



Results

Cleanroom samples were examined for total (living and dead) and viable (living only) microbial populations using molecular approaches and cultured isolates employing the traditional NASA standard spore assay (NSA), which predominantly isolated spores. The 130 NSA isolates were represented by 16 bacterial genera, of which 97% were identified as spore-formers via Sanger sequencing. The most spatially abundant isolate was Bacillus subtilis, and the most temporally abundant spore-former was Virgibacillus panthothenticus. The 16S rRNA gene-targeted amplicon sequencing detected 51 additional genera not found in the NSA method. The amplicon sequencing of the samples treated with propidium monoazide (PMA), which would differentiate between viable and dead organisms, revealed a total of 54 genera: 46 viable non-spore forming genera and 8 viable spore forming genera in these samples. The microbial diversity generated by the amplicon sequencing corresponded to ~86% non-spore-formers and ~14% spore-formers. The most common spatially distributed genera were Sphinigobium, Geobacillus, and Bacillus whereas temporally distributed common genera were Acinetobacter, Geobacilllus, and Bacillus. Single-cell genomics detected 6 genera in the sample analyzed, with the most prominent being Acinetobacter.



Conclusion

This study clearly established that detecting spores via NSA does not provide a complete assessment for the cleanliness of spacecraft-associated environments since it failed to detect several PP-relevant genera that were only recovered via molecular methods. This highlights the importance of a methodological paradigm shift to appropriately monitor bioburden in cleanrooms for not only the aeronautical industry but also for pharmaceutical, medical industries, etc., and the need to employ molecular sequencing to complement traditional culture-based assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{M2021,

title = {Genomic Variation Influences Methanothermococcus Fitness in Marine Hydrothermal Systems},

author = {Hoffert, M. and Anderson, R.E. and Reveillaud, J. and Murphy, L.G. and Stepanauskas, R. and Huber, J.A.},

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2021.714920/full},

doi = {https://doi.org/10.3389/fmicb.2021.714920},

year  = {2021},

date = {2021-08-20},

urldate = {2021-08-20},

journal = {Frontiers in Microbiology},

volume = {12},

pages = {714920},

abstract = {Hydrogenotrophic methanogens are ubiquitous chemoautotrophic archaea inhabiting globally distributed deep-sea hydrothermal vent ecosystems and associated subseafloor niches within the rocky subseafloor, yet little is known about how they adapt and diversify in these habitats. To determine genomic variation and selection pressure within methanogenic populations at vents, we examined five Methanothermococcus single cell amplified genomes (SAGs) in conjunction with 15 metagenomes and 10 metatranscriptomes from venting fluids at two geochemically distinct hydrothermal vent fields on the Mid-Cayman Rise in the Caribbean Sea. We observed that some Methanothermococcus lineages and their transcripts were more abundant than others in individual vent sites, indicating differential fitness among lineages. The relative abundances of lineages represented by SAGs in each of the samples matched phylogenetic relationships based on single-copy universal genes, and genes related to nitrogen fixation and the CRISPR/Cas immune system were among those differentiating the clades. Lineages possessing these genes were less abundant than those missing that genomic region. Overall, patterns in nucleotide variation indicated that the population dynamics of Methanothermococcus were not governed by clonal expansions or selective sweeps, at least in the habitats and sampling times included in this study. Together, our results show that although specific lineages of Methanothermococcus co-exist in these habitats, some outcompete others, and possession of accessory metabolic functions does not necessarily provide a fitness advantage in these habitats in all conditions. This work highlights the power of combining single-cell, metagenomic, and metatranscriptomic datasets to determine how evolution shapes microbial abundance and diversity in hydrothermal vent ecosystems.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Goordial2021,

title = {Microbial diversity and function in shallow subsurface sediment and oceanic lithosphere of the atlantis massif},

author = {Goordial, J. and D’angelo, T. and Labont\'{e}, J.M. and Poulton, N.J. and Brown, J.M. and Stepanauskas, R. and Fr\"{u}h-Green, G.L. and Orcutt, B.N.},

url = {https://journals.asm.org/doi/10.1128/mBio.00490-21},

doi = {https://doi.org/10.1128/mBio.00490-21 },

year  = {2021},

date = {2021-08-03},

journal = {mBio},

volume = {12},

number = {4},

pages = {e00490-21},

abstract = {The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm−3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (\<20 m below seafloor) subsurface of an actively serpentinizing marine system. The shallow subsurface biosphere of the Atlantis Massif was found to be distinct from communities observed in the nearby Lost City alkaline hydrothermal fluids and chimneys, yet similar to other low-temperature, aerobic subsurface settings. Genes associated with autotrophy were rare, although heterotrophy and aerobic carbon monoxide and formate cycling metabolisms were identified. Overall, this study reveals that the shallow subsurface of an oceanic core complex hosts a biosphere that is not fueled by active serpentinization reactions and by-products.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{\v{S}ul\v{c}ius2021,

title = {Exploring viral diversity in a gypsum karst lake ecosystem using targeted single-cell genomics},

author = {\v{S}ul\v{c}ius, S. and Alzbutas, G. and Juknevi\v{c}i\={u}t\.{e}, V. and \v{S}imoli\={u}nas, E. and Venckus, P. and \v{S}imoli\={u}nien\.{e}, M. and Pa\v{s}kauskas, R.},

url = {https://www.mdpi.com/2073-4425/12/6/886},

doi = {https://doi.org/10.3390/genes12060886},

year  = {2021},

date = {2021-06-08},

journal = {Genes},

volume = {12},

number = {6},

pages = {886},

abstract = {Little is known about the diversity and distribution of viruses infecting green sulfur bacteria (GSB) thriving in euxinic (sulfuric and anoxic) habitats, including gypsum karst lake ecosystems. In this study, we used targeted cell sorting combined with single-cell sequencing to gain insights into the gene content and genomic potential of viruses infecting sulfur-oxidizing bacteria Chlorobium clathratiforme, obtained from water samples collected during summer stratification in gypsum karst Lake Kirkilai (Lithuania). In total, 82 viral contigs were bioinformatically identified in 62 single amplified genomes (SAGs) of C. clathratiforme. The majority of viral gene and protein sequences showed little to no similarity with phage sequences in public databases, uncovering the vast diversity of previously undescribed GSB viruses. We observed a high level of lysogenization in the C. clathratiforme population, as 87% SAGs contained intact prophages. Among the thirty identified auxiliary metabolic genes (AMGs), two, thiosulfate sulfurtransferase (TST) and thioredoxin-dependent phosphoadenosine phosphosulfate (PAPS) reductase (cysH), were found to be involved in the oxidation of inorganic sulfur compounds, suggesting that viruses can influence the metabolism and cycling of this essential element. Finally, the analysis of CRISPR spacers retrieved from the consensus C. clathratiforme genome imply persistent and active virus\textendashhost interactions for several putative phages prevalent among C. clathratiforme SAGs. Overall, this study provides a glimpse into the diversity of phages associated with naturally occurring and highly abundant sulfur-oxidizing bacteria.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Murphy2021,

title = {Genomic analysis of the yet-uncultured binatota reveals broad methylotrophic, alkane-degradation, and pigment production capacities},

author = {Murphy, C.L. and Sheremet, A. and Dunfield, P.F. and Spear, J.R. and Stepanauskas, R. and Woyke, T. and Elshahed, M.S. and Youssef, N.H.},

url = {https://pubmed.ncbi.nlm.nih.gov/34006650/},

doi = {10.1128/mBio.00985-21},

year  = {2021},

date = {2021-05-18},

journal = {mBio},

volume = {12},

number = {3},

pages = {e00985-21},

abstract = {The recent leveraging of genome-resolved metagenomics has generated an enormous number of genomes from novel uncultured microbial lineages yet left many clades undescribed. Here, we present a global analysis of genomes belonging to Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. All orders in Binatota encoded the capacity for aerobic methylotrophy using methanol, methylamine, sulfomethanes, and chloromethanes as the substrates. Methylotrophy in Binatota was characterized by order-specific substrate degradation preferences, as well as extensive metabolic versatility, i.e., the utilization of diverse sets of genes, pathways, and combinations to achieve a specific metabolic goal. The genomes also encoded multiple alkane hydroxylases and monooxygenases, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids (lycopene, β- and γ-carotenes, xanthins, chlorobactenes, and spheroidenes) production. Further, the majority of genes involved in bacteriochlorophyll a, c, and d biosynthesis were identified, although absence of key genes and failure to identify a photosynthetic reaction center preclude proposing phototrophic capacities. Analysis of 16S rRNA databases showed the preferences of Binatota to terrestrial and freshwater ecosystems, hydrocarbon-rich habitats, and sponges, supporting their potential role in mitigating methanol and methane emissions, breakdown of alkanes, and their association with sponges. Our results expand the lists of methylotrophic, aerobic alkane-degrading, and pigment-producing lineages. We also highlight the consistent encountering of incomplete biosynthetic pathways in microbial genomes, a phenomenon necessitating careful assessment when assigning putative functions based on a set-threshold of pathway completion.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{WE2021,

title = {Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics},

author = {Kim WE and Charov K and Dzunkova M and Becraft ED and Brown J and Schulz F and Woyke T and La Clair JJ and Stepanauskas R and Burkart MD },

url = {https://pubs.acs.org/doi/full/10.1021/acschembio.1c00157},

year  = {2021},

date = {2021-05-06},

organization = {ACS Chem Biol},

abstract = {While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{S2021,

title = {A Genomic Catalogue of Earth’s Microbiomes},

author = {Nayfach S and Roux A and Seshadri R and Udwary D and Varghese N and Schulz F and Wu D and Paez-Espino D and Chen I-M and Huntemann M and Palaniappan K and Ladau J and Mukherjee S and Reddy TBK and Nielsen T and Kirton E and Faria EP and Edirisinghe JN and Henry CS and Jungbluth SP and Chivian D and Dehal P and Wood-Charlson EM and Arkin AP and Tringe S and Visel A and IMG/M Data Consortium and Woyke T and Mouncey NJ and Ivanova NN and Kyrpides NC and Eloe-Fadrosh EA},

doi = {https://doi.org/10.1038/s41587-020-0718-6},

year  = {2021},

date = {2021-04-12},

journal = {Nature Biotechnology},

volume = {39},

pages = {499-509},

abstract = {The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to \>10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ED2021,

title = {Evolutionary stasis of a deep subsurface microbial lineage},

author = {Becraft ED and Lau MCY and Bezuidt OKI and Brown JM and Labont\'{e} JM and Kauneckaite-Griguole K and Salkauskaite R and Alzbutas G and Sackett JD and Kruger BR and Kadnikov V and Van Heerden E and Moser D and Ravin N and Onstott T and Stepanauskas R },

url = {https://www.nature.com/articles/s41396-021-00965-3#citeas},

doi = {https://doi.org/10.1038/s41396-021-00965-3},

year  = {2021},

date = {2021-04-06},

journal = {The ISME Journal},

abstract = {Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared \>99.2% average nucleotide identity, low frequency of SNP’s, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{EM2021,

title = {Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation},

author = {Fones EM and Colman DR and Kraus EA and Stepanauskas R and Templeton AS and Spear JR and Boyd ES },

url = {https://www.nature.com/articles/s41396-020-00838-1#citeas},

doi = {https://doi.org/10.1038/s41396-020-00838-1},

year  = {2021},

date = {2021-04-01},

journal = {The ISME Journal},

volume = {15},

pages = {1121-1135},

abstract = {Metagenome assembled genomes (MAGs) and single amplified genomes (SAGs) affiliated with two distinct Methanobacterium lineages were recovered from subsurface fracture waters of the Samail Ophiolite, Sultanate of Oman. Lineage Type I was abundant in waters with circumneutral pH, whereas lineage Type II was abundant in hydrogen rich, hyperalkaline waters. Type I encoded proteins to couple hydrogen oxidation to CO2 reduction, typical of hydrogenotrophic methanogens. Surprisingly, Type II, which branched from the Type I lineage, lacked homologs of two key oxidative [NiFe]-hydrogenases. These functions were presumably replaced by formate dehydrogenases that oxidize formate to yield reductant and cytoplasmic CO2 via a pathway that was unique among characterized Methanobacteria, allowing cells to overcome CO2/oxidant limitation in high pH waters. This prediction was supported by microcosm-based radiotracer experiments that showed significant biological methane generation from formate, but not bicarbonate, in waters where the Type II lineage was detected in highest relative abundance. Phylogenetic analyses and variability in gene content suggested that recent and ongoing diversification of the Type II lineage was enabled by gene transfer, loss, and transposition. These data indicate that selection imposed by CO2/oxidant availability drove recent methanogen diversification into hyperalkaline waters that are heavily impacted by serpentinization.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{O2021,

title = {The cyanobacterium Prochlorococcus has divergent light-harvesting antennae and may have evolved in a low-oxygen ocean},

author = {Ulloa O and Henr\'{i}quez-Castillo C and Ram\'{i}rez-Flandes S and Plominsky AM and Murillo AA and Morgan-Lang C and Hallam SJ and Stepanauskas R },

url = {https://www.pnas.org/content/118/11/e2025638118},

doi = {https://doi.org/10.1073/pnas.2025638118},

issn = {1091-6490},

year  = {2021},

date = {2021-03-16},

journal = {PNAS},

volume = {118},

number = {11},

abstract = {Marine picocyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean, where they exert a profound influence on elemental cycling and energy flow. The use of transmembrane chlorophyll complexes instead of phycobilisomes as light-harvesting antennae is considered a defining attribute of Prochlorococcus. Its ecology and evolution are understood in terms of light, temperature, and nutrients. Here, we report single-cell genomic information on previously uncharacterized phylogenetic lineages of this genus from nutrient-rich anoxic waters of the eastern tropical North and South Pacific Ocean. The most basal lineages exhibit optical and genotypic properties of phycobilisome-containing cyanobacteria, indicating that the characteristic light-harvesting antenna of the group is not an ancestral attribute. Additionally, we found that all the indigenous lineages analyzed encode genes for pigment biosynthesis under oxygen-limited conditions, a trait shared with other freshwater and coastal marine cyanobacteria. Our findings thus suggest that Prochlorococcus diverged from other cyanobacteria under low-oxygen conditions before transitioning from phycobilisomes to transmembrane chlorophyll complexes and may have contributed to the oxidation of the ancient ocean.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JM2020,

title = {Single Cell Genomics Reveals Viruses Consumed by Marine Protists},

author = {Brown JM and Labont\'{e} JM and Brown J and Record NR and Poulton NJ and Sieracki ME and Logares R and Stepanauskas R},

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2020.524828/full},

doi = {https://doi.org/10.3389/fmicb.2020.524828},

year  = {2020},

date = {2020-09-24},

journal = {Frontiers in Microbiology},

volume = {11},

number = {524828},

abstract = {The predominant model of the role of viruses in the marine trophic web is that of the “viral shunt,” where viral infection funnels a substantial fraction of the microbial primary and secondary production back to the pool of dissolved organic matter. Here, we analyzed the composition of non-eukaryotic DNA associated with individual cells of small, planktonic protists in the Gulf of Maine (GoM) and the Mediterranean Sea. We found viral DNA associated with a substantial fraction cells from the GoM (51%) and the Mediterranean Sea (35%). While Mediterranean SAGs contained a larger proportion of cells containing bacterial sequences (49%), a smaller fraction of cells contained bacterial sequences in the GoM (19%). In GoM cells, nearly identical bacteriophage and ssDNA virus sequences where found across diverse lineages of protists, suggesting many of these viruses are non-infective. The fraction of cells containing viral DNA varied among protistan lineages and reached 100% in Picozoa and Choanozoa. These two groups also contained significantly higher numbers of viral sequences than other identified taxa. We consider mechanisms that may explain the presence of viral DNA in protistan cells and conclude that protistan predation on free viral particles contributed to the observed patterns. These findings confirm prior experiments with protistan isolates and indicate that the viral shunt is complemented by a viral link in the marine microbial food web. This link may constitute a sink of viral particles in the ocean and has implications for the flow of carbon through the microbial food web.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JP2020,

title = {Ancestral absence of electron transport chains in Patescibacteria and DPANN},

author = {Beam JP and Becraft ED and Brown JM and Schulz F and Jarett JK and Bezuidt O and Poulton NJ and Clark K and Dunfield PF and Ravin NV and Spear JR and Hedlund BP and Kormas KA and Sievert SM and Elshahed MS and Barton HA and Stott MB and Eisen JA and Moser DP and Onstott TC and Woyke T and Stepanauskas R},

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2020.01848/full},

doi = {https://doi.org/10.3389/fmicb.2020.01848},

year  = {2020},

date = {2020-08-17},

journal = {Frontiers in Microbiology},

volume = {11},

number = {1848},

abstract = {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\textendashcell 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JK2020,

title = {Insights into the dynamics between viruses and their hosts in a hot spring microbial mat},

author = {Jarett JK and D\v{z}unkov\'{a} M and Schulz F and Roux S and Paez-Espino D and Eloe-Fadrosh E and Jungbluth SP and Ivanova N and Spear JR and Carr SA and Trivedi CB and Corsetti FA and Johnson HA and Becraft E and Kyrpides N and Stepanauskas R and Woyke T},

url = {https://www.nature.com/articles/s41396-020-0705-4#citeas},

doi = {https://doi.org/10.1038/s41396-020-0705-4},

year  = {2020},

date = {2020-07-07},

journal = {The ISME Journal},

volume = {14},

pages = {2527-2541},

abstract = {Our current knowledge of host\textendashvirus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host\textendashvirus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus\textendashhost range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host\textendashvirus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed “Piggyback-the-Winner” theory.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ML2020,

title = {Hiding in Plain Sight: The Globally Distributed Bacterial Candidate Phylum PAUC34f},

author = {Chen ML and Becraft ED and Pachiadaki M and Brown JM and Jarett JK and Gasol JM and Ravin NV and Moser DP and Nunoura T and Herndl GJ and Woyke T and Stepanauskas R },

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2020.00376/full},

doi = {https://doi.org/10.3389/fmicb.2020.00376},

year  = {2020},

date = {2020-03-12},

journal = {Frontiers in Microbiology},

volume = {11},

number = {376},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JHW2020,

title = {Pangenomics Analysis Reveals Diversification of Enzyme Families and Niche Specialization in Globally Abundant SAR202 Bacteria},

author = {Saw JHW and Nunoura T and Hirai M and Takaki Y and Parsons R and Michelsen M and Longnecker K and Kujawinski EB and Stepanauskas R and Landry Z and Carlson CA and Giovannoni SJ },

url = {https://mbio.asm.org/content/11/1/e02975-19},

doi = {10.1128/mBio.02975-19},

year  = {2020},

date = {2020-01-07},

journal = {mBio},

volume = {11},

number = {1},

pages = {e02975-19},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{MG2019,

title = {Charting the complexity of the marine microbiome through single cell genomics},

author = {Pachiadaki MG and Brown JM and Brown J and Bezuidt O and Berube PM and Biller SJ and Poulton NJ and Burkart MD and La Clair JJ and Chisholm SW and Stepanauskas R},

url = {https://reader.elsevier.com/reader/sd/pii/S0092867419312735?token=5CCA0F9F7B2364A01F02747FA24E677BBD690529309015082F4BD56865547E22999EEFA8E287D658003474FFC76566E3},

doi = {https://doi.org/10.1016/j.cell.2019.11.017},

year  = {2019},

date = {2019-12-12},

journal = {Cell},

volume = {179},

number = {7},

pages = {1623-1635},

abstract = {Marine bacteria and archaea play key roles in global biogeochemistry. To improve our understanding of this complex microbiome, we employed single-cell genomics and a randomized, hypothesis-agnostic cell selection strategy to recover 12,715 partial genomes from the tropical and subtropical euphotic ocean. A substantial fraction of known prokaryoplankton coding potential was recovered from a single, 0.4 mL ocean sample, which indicates that genomic information disperses effectively across the globe. Yet, we found each genome to be unique, implying limited clonality within prokaryoplankton populations. Light harvesting and secondary metabolite biosynthetic pathways were numerous across lineages, highlighting the value of single-cell genomics to advance the identification of ecological roles and biotechnology potential of uncultured microbial groups. This genome collection enabled functional annotation and genus-level taxonomic assignments for \>80% of individual metagenome reads from the tropical and subtropical surface ocean, thus offering a model to improve reference genome databases for complex microbiomes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JM2019,

title = {Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system},

author = {Labont\'{e} JM and Pachiadaki M and Fergusson E and McNichol J and Grosche A and Gulmann LK and Vetriani C and Sievert SM and Stepanauskas R},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581674/},

doi = {10.3389/fmicb.2019.01262},

year  = {2019},

date = {2019-06-12},

journal = {Frontiers in Microbiology},

volume = {10},

pages = {1262},

abstract = {Phage\textendashhost 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\textendashhost 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{LR2019,

title = {Red Sea SAR11 and Prochlorococcus Single-Cell Genomes Reflect Globally Distributed Pangenomes},

author = {Thompson LR and Haroon MF and Shibl AA and Cahill MJ and Ngugi DK and Williams GJ and Morton JT and Knight R and Goodwin KD and Stingl U},

url = {https://aem.asm.org/content/85/13/e00369-19},

doi = {10.1128/AEM.00369-19},

year  = {2019},

date = {2019-04-26},

journal = {Applied and Environmental Microbiology},

volume = {85},

abstract = {Evidence suggests many marine bacteria are cosmopolitan, with widespread but sparse strains poised to seed abundant populations under conducive growth conditions. However, studies supporting this “microbial seed bank” hypothesis have analyzed taxonomic marker genes rather than whole genomes/metagenomes, leaving open the possibility that disparate ocean regions harbor endemic gene content. The Red Sea is isolated geographically from the rest of the ocean and has a combination of high irradiance, high temperature, and high salinity that is unique among the oceans; we therefore asked whether it harbors endemic gene content. We sequenced and assembled single-cell genomes of 21 SAR11 (subclades Ia, Ib, Id, and II) and 5 Prochlorococcus (ecotype HLII) samples from the Red Sea and combined them with globally sourced reference genomes to cluster genes into ortholog groups (OGs). Ordination of OG composition could distinguish clades, including phylogenetically cryptic Prochlorococcus ecotypes LLII and LLIII. Compared with reference genomes, 1% of Prochlorococcus and 17% of SAR11 OGs were unique to the Red Sea genomes (RS-OGs). Most (83%) RS-OGs had no annotated function, but 65% of RS-OGs were expressed in diel Red Sea metatranscriptomes, suggesting they are functional. Searching Tara Oceans metagenomes, RS-OGs were as likely to be found as non-RS-OGs; nevertheless, Red Sea and other warm samples could be distinguished from cooler samples using the relative abundances of OGs. The results suggest that the prevalence of OGs in these surface ocean bacteria is largely cosmopolitan, with differences in population metagenomes manifested by differences in relative abundance rather than complete presence/absence of OGs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{M2019b,

title = {Phylogenomics suggests oxygen availability as a driving force in Thaumarchaeota evolution},

author = {Ren M and Feng X and Huang Y and Wang H and Hu Z and Clingenpeel S and Swan BK and Fonseca MM and Posada D and Stepanauskas R and Hollibaugh JT and Foster PG and Woyke T and Luo H},

url = {https://www.nature.com/articles/s41396-019-0418-8},

doi = {https://doi.org/10.1038/s41396-019-0418-8},

year  = {2019},

date = {2019-04-25},

journal = {ISME},

volume = {13},

pages = {2150-2161},

abstract = {Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in marine and terrestrial habitats, playing a major role in the global nitrogen cycle. However, their evolutionary history remains unexplored, which limits our understanding of their adaptation mechanisms. Here, our comprehensive phylogenomic tree of Thaumarchaeota supports three sequential events: origin of AOA from terrestrial non-AOA ancestors, colonization of the shallow ocean, and expansion to the deep ocean. Careful molecular dating suggests that these events coincided with the Great Oxygenation Event around 2300 million years ago (Mya), and oxygenation of the shallow and deep ocean around 800 and 635\textendash560 Mya, respectively. The first transition was likely enabled by the gain of an aerobic pathway for energy production by ammonia oxidation and biosynthetic pathways for cobalamin and biotin that act as cofactors in aerobic metabolism. The first transition was also accompanied by the loss of dissimilatory nitrate and sulfate reduction, loss of oxygen-sensitive pyruvate oxidoreductase, which reduces pyruvate to acetyl-CoA, and loss of the Wood\textendashLjungdahl pathway for anaerobic carbon fixation. The second transition involved gain of a K+ transporter and of the biosynthetic pathway for ectoine, which may function as an osmoprotectant. The third transition was accompanied by the loss of the uvr system for repairing ultraviolet light-induced DNA lesions. We conclude that oxygen availability drove the terrestrial origin of AOA and their expansion to the photic and dark oceans, and that the stressors encountered during these events were partially overcome by gene acquisitions from Euryarchaeota and Bacteria, among other sources.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{ME2019,

title = {Single cell genomics yields a wide diversity of small planktonic protists across major ocean ecosystems},

author = {Sieracki ME and Poulton NJ and Jaillon O and Wincker P and de Vargas C and Rubinat-Ripoll L and Stepanauskas R and Logares R and Massana R},

url = {https://www.nature.com/articles/s41598-019-42487-1},

doi = {https://doi.org/10.1038/s41598-019-42487-1},

year  = {2019},

date = {2019-04-15},

journal = {Scientific Reports},

volume = {9},

pages = {6025},

abstract = {Marine planktonic protists are critical components of ocean ecosystems and are highly diverse. Molecular sequencing methods are being used to describe this diversity and reveal new associations and metabolisms that are important to how these ecosystems function. We describe here the use of the single cell genomics approach to sample and interrogate the diversity of the smaller (pico- and nano-sized) protists from a range of oceanic samples. We created over 900 single amplified genomes (SAGs) from 8 Tara Ocean samples across the Indian Ocean and the Mediterranean Sea. We show that flow cytometric sorting of single cells effectively distinguishes plastidic and aplastidic cell types that agree with our understanding of protist phylogeny. Yields of genomic DNA with PCR-identifiable 18S rRNA gene sequence from single cells was low (15% of aplastidic cell sorts, and 7% of plastidic sorts) and tests with alternate primers and comparisons to metabarcoding did not reveal phylogenetic bias in the major protist groups. There was little evidence of significant bias against or in favor of any phylogenetic group expected or known to be present. The four open ocean stations in the Indian Ocean had similar communities, despite ranging from 14°N to 20°S latitude, and they differed from the Mediterranean station. Single cell genomics of protists suggests that the taxonomic diversity of the dominant taxa found in only several hundreds of microliters of surface seawater is similar to that found in molecular surveys where liters of sample are filtered.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{NH2019,

title = {Genomic characterization of candidate division LCP-89 reveals an atypical cell wall structure, microcompartment production, and dual respiratory and fermentative capacities},

author = {Youssef NH and Farag IF and Hahn CR and Jarett J and Becraft E and Eloe-Fadrosh E and Lightfoot J and Bourgeois A and Cole T and Ferrante S and Truelock M and Marsh W and Jamaleddine M and Ricketts S and Simpson R and McFadden A and Hoff W and Ravin NV and Sievert S and Stepanauskas R and Woyke T and Elshahed M},

url = {https://aem.asm.org/content/85/10/e00110-19},

doi = {10.1128/AEM.00110-19},

year  = {2019},

date = {2019-03-22},

journal = {Applied and Environmental Microbiology},

volume = {85},

pages = {e00110-19},

abstract = {Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JD2019,

title = {Four draft single-cell genome sequences of novel, nearly identical Kiritimatiellaeota strains isolated from the continental deep subsurface},

author = {Sackett JD and Kruger BR and Becraft ED and Jarett JK and Stepanauskas R and Woyke T and Moser DP},

url = {https://mra.asm.org/content/8/11/e01249-18},

doi = {10.1128/MRA.01249-18},

year  = {2019},

date = {2019-03-14},

journal = {Microbiology Resource Announcements},

volume = {8},

pages = {e01249-18},

abstract = {The recently proposed bacterial phylum Kiritimatiellaeota represents a globally distributed monophyletic clade distinct from other members of the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum. Here, we present four phylogenetically distinct single-cell genome sequences from within the Kiritimatiellaeota lineage sampled from deep continental subsurface aquifer fluids of the Death Valley Regional Flow System in the United States.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{SA2019,

title = {Carboxydotrophy potential of uncultivated Hydrothermarchaeota from the subseafloor crustal biosphere},

author = {Carr SA and Jungbluth SP and Eloe-Fadrosh EA and Stepanauskas R and Woyke T and Rapp\'{e} MS and Orcutt BN},

url = {https://www.nature.com/articles/s41396-019-0352-9},

doi = {https://doi.org/10.1038/s41396-019-0352-9},

issn = {1751-7370},

year  = {2019},

date = {2019-02-07},

journal = {The ISME Journal},

volume = {13},

pages = {1457-1468},

abstract = {The exploration of Earth’s terrestrial subsurface biosphere has led to the discovery of several new archaeal lineages of evolutionary significance. Similarly, the deep subseafloor crustal biosphere also harbors many unique, uncultured archaeal taxa, including those belonging to Candidatus Hydrothermarchaeota, formerly known as Marine Benthic Group-E. Recently, Hydrothermarchaeota was identified as an abundant lineage of Juan de Fuca Ridge flank crustal fluids, suggesting its adaptation to this extreme environment. Through the investigation of single-cell and metagenome-assembled genomes, we provide insight into the lineage’s evolutionary history and metabolic potential. Phylogenomic analysis reveals the Hydrothermarchaeota to be an early-branching archaeal phylum, branching between the superphylum DPANN, Euryarchaeota, and Asgard lineages. Hydrothermarchaeota genomes suggest a potential for dissimilative and assimilative carbon monoxide oxidation (carboxydotrophy), as well as sulfate and nitrate reduction. There is also a prevalence of chemotaxis and motility genes, indicating adaptive strategies for this nutrient-limited fluid-rock environment. These findings provide the first genomic interpretations of the Hydrothermarchaeota phylum and highlight the anoxic, hot, deep marine crustal biosphere as an important habitat for understanding the evolution of early life.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{PM2019,

title = {Emergence of trait variability through the lens of nitrogen assimilation in Prochlorococcus},

author = {Berube PM and Rasmussen A and Braakman R and Stepanauskas R and Chisholm SW},

url = {https://elifesciences.org/articles/41043},

doi = {10.7554/eLife.41043},

year  = {2019},

date = {2019-02-01},

journal = {eLife},

volume = {8},

abstract = {Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages but occur at an intermediate frequency that is randomly distributed within recently emerged clades. The distribution of nitrate assimilation genes within clades appears largely governed by vertical inheritance, gene loss, and homologous recombination. By mapping this process onto a model of Prochlorococcus’ macroevolution, we propose that niche-constructing adaptive radiations and subsequent niche partitioning set the stage for loss of nitrate assimilation genes from basal lineages as they specialized to lower light levels. Retention of these genes in recently emerged lineages has likely been facilitated by selection as they sequentially partitioned into niches where nitrate assimilation conferred a fitness benefit.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Y2019,

title = {Genomics insights into ecotype formation of ammonia-oxidizing archaea in the deep ocean},

author = {Wang Y and Huang JM and Cui GJ and Nunoura T and Takaki Y and Li WL and Li J and Gao ZM and Takai K and Zhang AQ and Stepanauskas R},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.14518},

doi = {https://doi.org/10.1111/1462-2920.14518},

year  = {2019},

date = {2019-01-31},

journal = {Environmental Microbiology },

volume = {21},

number = {2},

pages = {716-729},

abstract = {Various lineages of ammonia‐oxidizing archaea (AOA) are present in deep waters, but the mechanisms that determine ecotype formation are obscure. We studied 18 high‐quality genomes of the marine group I AOA lineages (alpha, gamma and delta) from the Mariana and Ogasawara trenches. The genomes of alpha AOA resembled each other, while those of gamma and delta lineages were more divergent and had even undergone insertion of some phage genes. The instability of the gamma and delta AOA genomes could be partially due to the loss of DNA polymerase B (polB) and methyladenine DNA glycosylase (tag) genes responsible for the repair of point mutations. The alpha AOA genomes harbour genes encoding a thrombospondin‐like outer membrane structure that probably serves as a barrier to gene flow. Moreover, the gamma and alpha AOA lineages rely on vitamin B12‐independent MetE and B12‐dependent MetH, respectively, for methionine synthesis. The delta AOA genome contains genes involved in uptake of sugar and peptide perhaps for heterotrophic lifestyle. Our study provides insights into co‐occurrence of cladogenesis and anagenesis in the formation of AOA ecotypes that perform differently in nitrogen and carbon cycling in dark oceans.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{PB2019,

title = {Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria},

author = {Matheus Carnevali PB and Schulz F and Castelle CJ and Kantor RS and Shih PM and Sharon I and Santini JM and Olm MR and Amano Y and Thomas BC and Anantharaman K and Burstein D and Becraft ED and Stepanauskas R and Woyke T and Banfield JF},

url = {https://www.nature.com/articles/s41467-018-08246-y},

doi = {https://doi.org/10.1038/s41467-018-08246-y},

issn = {2041-1723},

year  = {2019},

date = {2019-01-28},

journal = {Nature Communications},

volume = {10},

number = {463},

abstract = {The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H2 metabolism were central metabolic features. The ability to use O2 as a terminal electron acceptor must have been subsequently acquired by these lineages.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{YM2018,

title = {Genomic insight into the predominance of candidate phylum Atribacteria JS1 lineage in marine sediments},

author = {Lee YM and Hwang K and Lee JI and Kim M and Hwang CY and Noh HJ and Choi H and Lee HK and Chun J and Hong SG and Shin SC },

url = {https://www.frontiersin.org/articles/10.3389/fmicb.2018.02909/full},

doi = { 10.3389/fmicb.2018.02909},

year  = {2018},

date = {2018-11-29},

journal = {Frontiers in Microbiology},

volume = {9},

abstract = {Candidate phylum Atribacteria JS1 lineage is one of the predominant bacterial groups in anoxic subseafloor sediments, especially in organic-rich or gas hydrate-containing sediments. However, due to the lack of axenic culture representatives, metabolic potential and biogeochemical roles of this phylum have remained elusive. Here, we examined the microbial communities of marine sediments of the Ross Sea, Antarctica, and found candidate phylum Atribacteria JS1 lineage was the most abundant candidate phylum accounting for 9.8\textendash40.8% of the bacterial communities with a single dominant operational taxonomic unit (OTU). To elucidate the metabolic potential and ecological function of this species, we applied a single-cell genomic approach and obtained 18 single-cell amplified genomes presumably from a single species that was consistent with the dominant OTU throughout the sediments. The composite genome constructed by co-assembly showed the highest genome completeness among available Atribacteria JS1 genomes. Metabolic reconstruction suggested fermentative potential using various substrates and syntrophic acetate oxidation coupled with hydrogen or formate scavenging methanogens. This metabolic potential supports the predominance of Atribacteria JS1 in anoxic environments expanding our knowledge of the ecological function of this uncultivated group.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{JK2018,

title = {Single-cell genomics of co-sorted Nanoarchaeota suggests novel putative host associations and diversification of proteins involved in symbiosis},

author = {Jarett JK and Nayfach S and Podar M and Inskeep W and Ivanova NN and Munson-Mcgee J and Schulz F and Young M and Jay ZJ and Beam JP and Kyrpides NC and Malmstrom RR and Stepanauskas R and Woyke T},

url = {https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0539-8},

doi = {https://doi.org/10.1186/s40168-018-0539-8},

issn = {2049-2618},

year  = {2018},

date = {2018-09-17},

journal = {Microbiome},

volume = {6},

number = {161},

abstract = {Nanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{PM2018,

title = {Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments},

author = {Berube PM and Biller SJ and Hackl T and Hogle SL and Satinsky BM and Becker JW and Braakman R and Collins SB and Kelly L and Berta-Thompson J and Coe A and Bergauer K and Bouman HA and Browning TJ and De Corte D and Hassler C and Hulata Y and Jacquot JE and Maas EW and Reinthaler T and Sintes E and Yokokawa T and Lindell D and Stepanauskas R and Chisholm SW},

url = {https://www.nature.com/articles/sdata2018154},

doi = {https://doi.org/10.1038/sdata.2018.154},

issn = {2052-4463},

year  = {2018},

date = {2018-09-04},

journal = {Scientific Data},

volume = {5},

abstract = {Prochlorococcus and Synechococcus are the dominant primary producers in marine ecosystems and perform a significant fraction of ocean carbon fixation. These cyanobacteria interact with a diverse microbial community that coexists with them. Comparative genomics of cultivated isolates has helped address questions regarding patterns of evolution and diversity among microbes, but the fraction that can be cultivated is miniscule compared to the diversity in the wild. To further probe the diversity of these groups and extend the utility of reference sequence databases, we report a data set of single cell genomes for 489 Prochlorococcus, 50 Synechococcus, 9 extracellular virus particles, and 190 additional microorganisms from a diverse range of bacterial, archaeal, and viral groups. Many of these uncultivated single cell genomes are derived from samples obtained on GEOTRACES cruises and at well-studied oceanographic stations, each with extensive suites of physical, chemical, and biological measurements. The genomic data reported here greatly increases the number of available Prochlorococcus genomes and will facilitate studies on evolutionary biology, microbial ecology, and biological oceanography.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}