Biology subjects

Stepanauskas, R.

Publications and source records attributed to Stepanauskas, R..

14 recordsLinked to original sources

bioRxiv2026

Global, single-cell-resolution of antiviral defenses in marine prokaryoplankton

Background Growing evidence suggests that variation in resistance to viral infections by marine prokaryoplankton enables the coexistence of both viral and host populations. Recent experimental work on model organisms has revealed dozens of novel antiviral defense mechanisms. This diversity has prompted the hypothesis of the pan-immunity model, in which diverse defenses are a shared resource among closely related individuals to broaden the population's resistance and minimize an individual's burden. The composition and abundance of such defense pools in natural microbial communities, however, remain largely unknown. Here, we begin to parameterize resistance in marine prokaryotes and define their defense repertoire by quantifying antiviral defenses across thousands of randomized single-amplified genomes (SAGs) from a global collection of seawater samples. Results Prokaryoplankton SAGs contained an average of 1.1 defenses, with dark ocean (200 m - 11 km) prokaryotes having a slightly higher genomic load of defenses as compared to the sunlit, surface ocean. The number of defenses per cell was taxon-specific, with little or no relationship to depth, taxon abundance, estimated maximal growth rate, or viral infection rate. The most numerous and taxonomically widespread defenses were restriction modification systems, followed by dGTPase (mostly in Pelagibacterales) in the sunlit ocean and AbiU (mostly in Nitrososphaerales) in the dark ocean. Most other defense types were rare (< 0.1% SAGs) yet distributed across distant taxa (> 3 phyla). We found evidence for the cross-domain exchange of the most common defense system, RM II, between archaea and bacteria, which improves our understanding of the biology of this prevalent defense and could be consequential in predicting target motifs for connecting viruses with potential hosts in epigenetic studies. Conclusions Collectively, these results suggest lineages restrict genomic real estate for defenses yet enable extensive lateral transfer, potentially for the maintenance of defense variation at the community level rather than only among closely related individuals. This study provides a quantitative atlas of prokaryoplankton immunity toward grounding our understanding of virus-microbe interactions in the Earth's largest biome, the open ocean.

microbiology↗

bioRxiv2026

Single-cell-resolved frequency and modes of phage interactions with prokaryoplankton in the tropical surface ocean

Marine planktonic viruses play critical roles in shaping microbial communities and driving global biogeochemical cycles. However, quantitative, microbiome-wide analyses of marine prokaryoplankton-virus interactions in situ and their ecological impacts remain challenging due to the vast diversity of viral genomes and interaction modes and the limitations of existing methodologies. Here, we utilized GORG-Tropics, a global collection of 12,715 single amplified genomes (SAGs) generated from randomly sampled marine prokaryoplankton cells, to determine the frequency and modes of their interactions with viruses in the tropical surface ocean. We found 4.2% (range 1%-19% among samples) of GORG-Tropics SAGs to contain phage genomic material, with the highest frequency found in productive ocean regions. Prokaryoplankton lineages known to have high metabolic rates, including Prochlorococcus and Rhodobacterales, had a substantially larger fraction of cells associated with viruses (10-12% of SAGs) as compared to the less active but highly abundant lineages including Pelagibacterales (2.2% of SAGs). Cell-virus associations indicative of lysogeny were elevated in Alphaproteobacteria relative to other taxa. The collection of phages recovered from individual SAGs exhibited genomic diversity that bridged order-level taxonomies, indicating high diversity and genomic connectivity within wild phage populations. A substantial fraction of the observed cell-virus associations disagreed with the computationally predicted host identity of the virus, indicative of non-infective interactions. The extent of genetic exchange across tailed bacteriophages infecting different hosts, and connected to taxonomically distant phages provided further evidence for the role of non-infective phage entry in the lateral transfer between tailed phages. This study provides large-scale quantitative evidence of viral infection rates in the collective prokaryoplankton community across the global surface tropical ocean through large-scale identification and quantification of the specific phages, hosts, and modes of interaction at the resolution of individual cells. Our results confirm prior reports on the overall frequency of prokaryoplankton infections with viruses in the oligotrophic tropical surface ocean. Our findings uncover non-infective phage-cell associations that may be contributing to the lateral transfer of viral genes and the nutrition of marine prokaryoplankton.

microbiology↗

bioRxiv2025

Global Metagenomics Reveals Hidden Protist Diversity

Protists, defined as eukaryotes distinct from animals, plants, and fungi, are a polyphyletic group that dominates the eukaryotic tree of life, exhibiting significant phylogenetic diversity and fulfilling critical ecological roles. Historically, research has prioritized protists associated with animals and plants, particularly those of medical significance, thereby overlooking the majority of protist diversity. Conventional molecular methods, such as 18S rRNA gene amplicon sequencing, frequently encounter limitations, including primer binding bias and PCR bias caused by gene length variations, resulting in a biased representation of protistan diversity. Further, most protist lineages are notoriously difficult to cultivate. Here, we apply a cultivation-independent approach in which we analyzed over 30,000 assembled metagenomes and protist single cell genomes and 21 long 18S rRNA gene amplicon data sets from various global ecosystems, including marine, freshwater, and soil environments. We recovered 157,956 18S rRNA gene sequences ([≥]800 bp), which clustered into 103,338 operational taxonomic units (OTUs) at 97% sequence identity and 24,438 OTUs at 85% identity. Notably, 81% of 9,543 non-singleton clusters at 85% identity classified as protists consisted exclusively of environmental sequences, uncovering a wealth of novel, uncultivated, and unclassified protist diversity. A comprehensive taxonomic framework of eukaryotes based on concatenated 18S and 28S rRNA genes that incorporated most novel lineages revealed substantial underrepresentation of Amoebozoa, Discoba, and Rhizaria in reference databases, with many lacking isolate or genome sequence representation. Further, we identified 13 eukaryotic lineages with novelty on higher taxonomic ranks, such as class and phylum-level, that lack representation in public databases. The corresponding 85% OTUs were primarily affiliated with Excavata, with some branching deeply in the eukaryotic tree. Comprehensive analysis of the global distribution of protists revealed uneven microbial diversity across supergroups and ecosystems, with notable novelty particularly in soil and marine environments. We then examined co-occurrence between protists and prokaryotes, predicting putative symbiotic or predator-prey relationships, particularly among understudied protist groups with bacteria such as Verrucomicrobia and Rickettsiales. Our results substantially enhance the understanding of protistan diversity and distribution, revealing taxonomic blind spots and laying groundwork for future studies of these organisms ecological roles.

microbiology↗

bioRxiv2023

Flotillin-Associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes

Microbial rhodopsins are often found more than once in a single genome (paralogs) that 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). They are quite divergent from the other proteorhodopsin paralog and contain either DTT, DTL or DNI motives 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 their relatives in freshwater metagenomes that lack this key amino acid. Alfa-fold predictions of marine FArhodopsins indicate that their retinal pocket might be very reduced or absent, hinting that they are retinal-less (blind). Freshwater FArhodopsins were more diverse than marine FArhodopsins, but we could not determine if they are present as paralogs of other rhodopsins, 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. IMPORTANCERhodopsins 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 co-existing proteorhodopsin proton pumps. The absence or reduction of the retinal binding pocket points to drastically different physiology. In addition to their ecological importance, novel rhodopsins have biotechnological potential in the nascent field of optogenetics.

microbiology↗

bioRxiv2021

Single cell genome sequencing of laboratory mouse microbiota improves taxonomic and functional resolution of this model microbial community

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.

microbiology↗

bioRxiv2021

Strain-level variation influences Methanothermococcus fitness in marine hydrothermal systems

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 strains and their transcripts were more abundant than others in individual vent sites, and among the genes differentiating the Methanothermococcus SAGs were those related to nitrogen fixation and the CRISPR/Cas immune system. SAGs 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.

microbiology↗

bioRxiv2020

Novel integrative elements and genomic plasticity in ocean ecosystems

Horizontal gene transfer accelerates microbial evolution, promoting diversification and adaptation. The globally abundant marine cyanobacterium Prochlorococcus has a highly streamlined genome with frequent gene exchange reflected in its extensive pangenome. The source of its genomic variability, however, remains elusive since most cells lack the common mechanisms that enable horizontal gene transfer, including conjugation, transformation, plasmids and prophages. Examining 623 genomes, we reveal a diverse system of mobile genetic elements - cargo-carrying transposons we named tycheposons - that shape Prochlorococcus genomic plasticity. The excision and integration of tycheposons at seven tRNA genes drive the remodeling of larger genomic islands containing most of Prochlorococcus flexible genes. Most tycheposons carry genes important for niche differentiation through nutrient acquisition; others appear similar to phage parasites. Tycheposons are highly enriched in extracellular vesicles and phage particles in ocean samples, suggesting efficient routes for their dispersal, transmission and propagation. Supported by evidence for similar elements in other marine microbes, our work underpins the role of vesicle- and virus-mediated transfer of mobile genetic elements in the diversification and adaptation of microbes in dilute aquatic environments - adding a significant piece to the puzzle of what governs microbial evolution in the planets largest habitat.

microbiology↗

bioRxiv2020

Methylotrophy, alkane-degradation, and pigment production as defining features of the globally distributed yet-uncultured phylum Binatota

The recent leveraging of genome-resolved metagenomics has opened a treasure trove of genomes from novel uncultured microbial lineages, yet left many clades undescribed. We here present a global analysis of genomes belonging to the Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. All orders in the Binatota encoded the capacity for aerobic methylotrophy using methanol, methylamine, sulfomethanes, chloromethanes, and potentially methane as substrates. Methylotrophy in the 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 an arsenal of 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, {beta} and {gamma} 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 precludes proposing phototrophic capacities. Analysis of 16S rRNA databases showed Binatotas preferences to terrestrial and freshwater ecosystems, hydrocarbon-rich habitats, and sponges supporting their suggested potential role in mitigating methanol and methane emissions, alkanes degradation, and nutritional symbiosis 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 and gene shrapnel in microbial genomes, a phenomenon necessitating careful assessment when assigning putative functions based on a set-threshold of pathway completion.

microbiology↗

bioRxiv2020

Gene exchange networks define species-like units in marine prokaryotes

Post-submission note. Since the original submission of this manuscript to bioRxiv, we discovered that some of our results may be impacted by the limitations of some of the comparative genomics tools used in this study. We are working on a revised version of the manuscript. Although horizontal gene transfer is recognized as a major evolutionary process in Bacteria and Archaea, its general patterns remain elusive, due to difficulties tracking genes at relevant resolution and scale within complex microbiomes. To circumvent these challenges, we analyzed a randomized sample of >12,000 genomes of individual cells of Bacteria and Archaea in the tropical and subtropical ocean - a well-mixed, global environment. We found that marine microorganisms form gene exchange networks (GENs) within which transfers of both flexible and core genes are frequent, including the rRNA operon that is commonly used as a conservative taxonomic marker. The data revealed efficient gene exchange among genomes with <28% nucleotide difference, indicating that GENs are much broader lineages than the nominal microbial species, which are currently delineated at 4-6% nucleotide difference. The 42 largest GENs accounted for 90% of cells in the tropical ocean microbiome. Frequent gene exchange within GENs helps explain how marine microorganisms maintain millions of rare genes and adapt to a dynamic environment despite extreme genome streamlining of their individual cells. Our study suggests that sharing of pangenomes through horizontal gene transfer is a defining feature of fundamental evolutionary units in marine planktonic microorganisms and, potentially, other microbiomes.

microbiology↗

bioRxiv2020

Ancestral absence of electron transport chains in Patescibacteria and DPANN

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of minimal cellular energy transduction mechanisms that potentially precede the evolution of respiration, thus relying solely on fermentation for energy conservation.

evolutionary biology↗

bioRxiv2019

Pangenomics reveal diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria

It has been hypothesized that abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolism 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 metabolic schemes proposed, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore this question, we reconstructed SAR202 single amplified genomes and metagenome-assembled genomes from locations around the world, including the deepest ocean trenches. In analyses of 122 SAR202 genomes that included six subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, and further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the Group III SAR202, in the proposed class Monstramaria and expansions of dioxygenase enzymes were prevalent in Group IV. We found that Group I SAR202 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 fluorescence in situ hybridization 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 in stratified water columns.\n\nImportanceThe oceans contain an estimated 662 Pg C of dissolved organic carbon (DOC). Information about microbial interactions with this vast resource is limited, despite broad recognition that DOM turnover has a major impact on the global carbon cycle. To explain patterns in the genomes of marine bacteria we propose hypothetical metabolic pathways for the oxidation of organic molecules that are resistant to oxidation via common pathways. The hypothetical schemes we propose suggest new metabolism and classes of compounds that could be important for understanding of the distribution of organic carbon throughout the biosphere. These genome-based schemes will remain hypothetical until evidence from experimental cell biology can be gathered to test them, but until then they provide a perspective that directs our attention to the biochemistry of resistant DOM metabolism. Our findings also fundamentally change our understanding of the ecology of SAR202, showing that metabolically diverse variants of these cells occupy niches spanning all depths, and are not relegated to the dark ocean.

genomics↗

bioRxiv2018

Emergence of trait variability through the lens of nitrogen assimilation in Prochlorococcus

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. The marine cyanobacterium Prochlorococcus is a useful model system for understanding how trait variability emerges within microbial species: Its functional diversity is overlaid on measurable environmental gradients, providing a powerful lens into large-scale evolutionary processes. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing the allocation of ecologically important functions within microbial species. We find that nitrate assimilation genes are absent in basal lineages of Prochlorococcus 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, stochastic gene loss, and homologous recombination among closely related cells. 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.

evolutionary biology↗

bioRxiv2018

Hydrogen-based metabolism - An ancestral trait in lineages sibling to the Cyanobacteria

The metabolic machinery from which microbial aerobic respiration evolved is tightly linked to the origins of oxygenic Cyanobacteria (Oxyphotobacteria). Even though the majority of Oxyphotobacteria are photoautotrophs and can use carbohydrates with oxygen (O2) as the electron acceptor, all are fermenters under dark anoxic conditions. Studies suggest that the ancestor of Oxyphotobacteria may have used hydrogen (H2) as an electron donor and that two types of NiFe hydrogenases are essential for its oxidation. Melainabacteria and Sericytochromatia, close phylogenetic neighbors to Oxyphotobacteria comprise fermentative and aerobic representatives, or organisms capable of both. Margulisbacteria (candidate divisions RBX-1 and ZB3) and Saganbacteria (candidate division WOR-1), a novel cluster of bacteria phylogenetically related to Melainabacteria, Sericytochromatia and Oxyphotobacteria may further constrain the metabolic platform in which oxygenic photosynthesis and aerobic respiration arose. Here, we predict the metabolisms of Margulisbacteria and Saganbacteria from new and published metagenome-assembled genomes (MAGs) and single amplified genomes (SAGs), and compare them to their phylogenetic neighbors. Sediment-associated Margulisbacteria are predicted to have a fermentation-based metabolism featuring a variety of hydrogenases, a nitrogenase for nitrogen (N2) fixation, and electron bifurcating complexes involved in cycling of ferredoxin and NAD(P)H. Overall, the genomic features suggest the capacity for metabolic fine-tuning under strictly anoxic conditions. In contrast, the genomes of Margulisbacteria from the ocean ecosystem encode an electron transport chain that supports aerobic growth. Similarly, some Saganbacteria genomes encode various hydrogenases, and others may have the ability to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Like Melainabacteria and Sericytochromatia, Margulisbacteria and Saganbacteria have diverse energy metabolisms capable of fermentation, and aerobic or anaerobic respiration. In summary, our findings support the hypothesis that the ancestor of these groups was an anaerobe in which fermentation and H2 metabolism were central metabolic features. Our genomic data also suggests that contemporary lineages sibling to the Oxyphotobacteria may have acquired the ability to use O2 as a terminal electron acceptor under certain environmental conditions.

microbiology↗

bioRxiv2017

Solagigasbacteria: Lone genomic giants among the uncultured bacterial phyla

Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the \"Candidate phylum SPAM\" represents an interesting exception, by having large genomes (6-8 Mbps), high GC content (66%-71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual SPAM cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior metagenomic studies. Based on these observations, we propose renaming SPAM to \"Candidate phylum Solagigasbacteria\". Current evidence suggests that Solagigasbacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers and the deep subsurface, with no reports from marine environments to date.

microbiology↗