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Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters., Competing Interests: CG and VGP are co-inventors on a patent related to this work. CG and VGP are equity holders of BioSkryb Genomics. CG is a co-founder and Board member of BioSkryb Genomics. The remaining authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant-fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions., (© 2024. The Author(s).)

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing eight new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nano-scale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal non-canonical amino acid tagging (BONCAT) we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle., Competing Interests: Competing interest statement: none declared

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Villalobos-Escobedo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg , an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, "who is eating what?" and "who is active?" within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism's taxonomic identity and genome composition while providing quantitative estimates of the microorganism's isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes., Competing Interests: The authors declare no conflict of interest.

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Trexler, Van Goethem, Goudeau, Nath, Malmstrom, Northen and Couradeau.)

Bacterial species often undergo rampant recombination yet maintain cohesive genomic identity. Ecological differences can generate recombination barriers between species and sustain genomic clusters in the short term. But can these forces prevent genomic mixing during long-term coevolution? Cyanobacteria in Yellowstone hot springs comprise several diverse species that have coevolved for hundreds of thousands of years, providing a rare natural experiment. By analyzing more than 300 single-cell genomes, we show that despite each species forming a distinct genomic cluster, much of the diversity within species is the result of hybridization driven by selection, which has mixed their ancestral genotypes. This widespread mixing is contrary to the prevailing view that ecological barriers can maintain cohesive bacterial species and highlights the importance of hybridization as a source of genomic diversity., Competing Interests: Competing interests: The authors declare no competing interests.

Background: Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling., Results: Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10-33 atom% 13 C), even though the soils' overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera., Conclusions: Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract., (© 2022. The Author(s).)

Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor. We analyse complete 'Comchoano' genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella's that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs., (© 2022. The Author(s).)

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Candidatus ( Ca. ) Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells. These features, along with compartmentalization of genomic material and ribosomes in translationally active organelles bound by bioenergetic membranes, indicate gain of complexity in the Thiomargarita lineage and challenge traditional concepts of bacterial cells.

Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (" Candidatus Methanocomedenaceae") and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides , Anaerolineales , and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. IMPORTANCE Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

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., (© 2021. The Author(s).)

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study-subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes-offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon., (© 2022. The Author(s).)

Background: Oceanic microbiomes play a pivotal role in the global carbon cycle and are central to the transformation and recycling of carbon and energy in the ocean's interior. SAR324 is a ubiquitous but poorly understood uncultivated clade of Deltaproteobacteria that inhabits the entire water column, from ocean surface waters to its deep interior. Although some progress has been made in elucidating potential metabolic traits of SAR324 in the dark ocean, very little is known about the ecology and the metabolic capabilities of this group in the euphotic and twilight zones. To investigate the comparative genomics, ecology, and physiological potential of the SAR324 clade, we examined the distribution and variability of key genomic features and metabolic pathways in this group from surface waters to the abyss in the North Pacific Subtropical Gyre, one of the largest biomes on Earth., Results: We leveraged a pangenomic ecological approach, combining spatio-temporally resolved single-amplified genome, metagenomic, and metatranscriptomic datasets. The data revealed substantial genomic diversity throughout the SAR324 clade, with distinct depth and temporal distributions that clearly differentiated ecotypes. Phylogenomic subclade delineation, environmental distributions, genomic feature similarities, and metabolic capacities revealed strong congruence. The four SAR324 ecotypes delineated in this study revealed striking divergence from one another with respect to their habitat-specific metabolic potentials. The ecotypes living in the dark or twilight oceans shared genomic features and metabolic capabilities consistent with a sulfur-based chemolithoautotrophic lifestyle. In contrast, those inhabiting the sunlit ocean displayed higher plasticity energy-related metabolic pathways, supporting a presumptive photoheterotrophic lifestyle. In epipelagic SAR324 ecotypes, we observed the presence of two types of proton-pumping rhodopsins, as well as genomic, transcriptomic, and ecological evidence for active photoheterotrophy, based on xanthorhodopsin-like light-harvesting proteins., Conclusions: Combining pangenomic and both metagenomic and metatranscriptomic profiling revealed a striking divergence in the vertical distribution, genomic composition, metabolic potential, and predicted lifestyle strategies of geographically co-located members of the SAR324 bacterial clade. The results highlight the utility of metapangenomic approaches employed across environmental gradients, to decipher the properties and variation in function and ecological traits of specific phylogenetic clades within complex microbiomes. Video abstract., (© 2021. The Author(s).)

Temperate phages are viruses of bacteria that can establish two types of infection: a lysogenic infection in which the virus replicates with the host cell without producing virions, and a lytic infection where the host cell is eventually destroyed, and new virions are released. While both lytic and lysogenic infections are routinely observed in the environment, the ecological and evolutionary processes regulating these viral dynamics are still not well understood, especially for uncultivated virus-host pairs. Here, we characterized the long-term dynamics of uncultivated viruses infecting green sulfur bacteria (GSB) in a model freshwater lake (Trout Bog Lake, TBL). As no GSB virus has been formally described yet, we first used two complementary approaches to identify new GSB viruses from TBL; one in vitro based on flow cytometry cell sorting, the other in silico based on CRISPR spacer sequences. We then took advantage of existing TBL metagenomes covering the 2005-2018 period to examine the interactions between GSB and their viruses across years and seasons. From our data, GSB populations in TBL were constantly associated with at least 2-8 viruses each, including both lytic and temperate phages. The dominant GSB population in particular was consistently associated with two prophages with a nearly 100% infection rate for >10 years. We illustrate with a theoretical model that such an interaction can be stable given a low, but persistent, level of prophage induction in low-diversity host populations. Overall, our data suggest that lytic and lysogenic viruses can readily co-infect the same host population, and that host strain-level diversity might be an important factor controlling virus-host dynamics including lytic/lysogeny switch.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 10 8 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Quantitative stable isotope probing (qSIP) estimates isotope tracer incorporation into DNA of individual microbes and can link microbial biodiversity and biogeochemistry in complex communities. As with any quantitative estimation technique, qSIP involves measurement error, and a fuller understanding of error, precision, and statistical power benefits qSIP experimental design and data interpretation. We used several qSIP data sets-from soil and seawater microbiomes-to evaluate how variance in isotope incorporation estimates depends on organism abundance and resolution of the density fractionation scheme. We assessed statistical power for replicated qSIP studies, plus sensitivity and specificity for unreplicated designs. As a taxon's abundance increases, the variance of its weighted mean density declines. Nine fractions appear to be a reasonable trade-off between cost and precision for most qSIP applications. Increasing the number of density fractions beyond that reduces variance, although the magnitude of this benefit declines with additional fractions. Our analysis suggests that, if a taxon has an isotope enrichment of 10 atom% excess, there is a 60% chance that this will be detected as significantly different from zero (with alpha 0.1). With five replicates, isotope enrichment of 5 atom% could be detected with power (0.6) and alpha (0.1). Finally, we illustrate the importance of internal standards, which can help to calibrate per sample conversions of %GC to mean weighted density. These results should benefit researchers designing future SIP experiments and provide a useful reference for metagenomic SIP applications where both financial and computational limitations constrain experimental scope. IMPORTANCE One of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about their genomic content and potential functions but may not reflect an organism's activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions but often fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and functional activity of specific microbes within a naturally occurring community. Here, we explore how the resolution of density gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional experimental replication, and discuss cost-benefit balanced scenarios for SIP experimental design.

The rhizosphere is a hotspot for microbial carbon transformations, and is the entry point for root polysaccharides and polymeric carbohydrates that are important precursors to soil organic matter (SOM). However, the ecological mechanisms that underpin rhizosphere carbohydrate depolymerization are poorly understood. Using Avena fatua, a common annual grass, we analyzed time-resolved metatranscriptomes to compare microbial functions in rhizosphere, detritusphere, and combined rhizosphere-detritusphere habitats. Transcripts were binned using a unique reference database generated from soil isolate genomes, single-cell amplified genomes, metagenomes, and stable isotope probing metagenomes. While soil habitat significantly affected both community composition and overall gene expression, the succession of microbial functions occurred at a faster time scale than compositional changes. Using hierarchical clustering of upregulated decomposition genes, we identified four distinct microbial guilds populated by taxa whose functional succession patterns suggest specialization for substrates provided by fresh growing roots, decaying root detritus, the combination of live and decaying root biomass, or aging root material. Carbohydrate depolymerization genes were consistently upregulated in the rhizosphere, and both taxonomic and functional diversity were highest in the combined rhizosphere-detritusphere, suggesting coexistence of rhizosphere guilds is facilitated by niche differentiation. Metatranscriptome-defined guilds provide a framework to model rhizosphere succession and its consequences for soil carbon cycling.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.

Background: Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses., Methods: Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity., Results: Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade., Conclusions: With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirR DTS ) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirR DTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.

Exploration of environmental microbiomes has shed light on the ecological and evolutionary principles at play in natural ecosystems and has been further accelerated through the reconstruction of population genomes to provide genome-centric context. Yet technical challenges with traditional shotgun metagenomics remain for computationally intense short-read assembly, strain heterogeneity within communities, and depth of coverage required for low-abundance microbes. In this Perspective, we highlight three main avenues for promising future developments, including coupling stable isotope probing and genome-resolved metagenomics, applying fluorescence-activated cell sorting approaches to target mini-metagenomes within a larger community, and utilizing single-molecule long-read and synthetic long-read technology to link mobile elements to host microbial cells. These developments on the horizon will undoubtedly advance genome-resolved metagenomic approaches and enable a better understanding of uncultivated microbes in their natural environments., Competing Interests: Conflict of Interest Disclosures: R.R.M. has nothing to disclose. E.A.E.-F. has nothing to disclose., (Copyright © 2019 Malmstrom and Eloe-Fadrosh.)

Background: Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes., Methods: Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10 kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes., Results: Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥10 kb by 10 to 100-fold for low input metagenomes., Conclusions: PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes., Competing Interests: The authors declare there are no competing interests.

Using genome-wide mutant fitness assays in diverse bacteria, we identified novel oxidative pathways for the catabolism of 2-deoxy-d-ribose and 2-deoxy-d-ribonate. We propose that deoxyribose is oxidized to deoxyribonate, oxidized to ketodeoxyribonate, and cleaved to acetyl coenzyme A (acetyl-CoA) and glyceryl-CoA. We have genetic evidence for this pathway in three genera of bacteria, and we confirmed the oxidation of deoxyribose to ketodeoxyribonate in vitro . In Pseudomonas simiae, the expression of enzymes in the pathway is induced by deoxyribose or deoxyribonate, while in Paraburkholderia bryophila and in Burkholderia phytofirmans, the pathway proceeds in parallel with the known deoxyribose 5-phosphate aldolase pathway. We identified another oxidative pathway for the catabolism of deoxyribonate, with acyl-CoA intermediates, in Klebsiella michiganensis. Of these four bacteria, only P. simiae relies entirely on an oxidative pathway to consume deoxyribose. The deoxyribose dehydrogenase of P. simiae is either nonspecific or evolved recently, as this enzyme is very similar to a novel vanillin dehydrogenase from Pseudomonas putida that we identified. So, we propose that these oxidative pathways evolved primarily to consume deoxyribonate, which is a waste product of metabolism. IMPORTANCE Deoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not contain any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants' abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of metabolism. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

Although microbes mediate much of the biogeochemical cycling in freshwater, the categories of carbon and nutrients currently used in models of freshwater biogeochemical cycling are too broad to be relevant on a microbial scale. One way to improve these models is to incorporate microbial data. Here, we analyze both genes and genomes from three metagenomic time series and propose specific roles for microbial taxa in freshwater biogeochemical cycles. Our metagenomic time series span multiple years and originate from a eutrophic lake (Lake Mendota) and a humic lake (Trout Bog Lake) with contrasting water chemistry. Our analysis highlights the role of polyamines in the nitrogen cycle, the diversity of diazotrophs between lake types, the balance of assimilatory vs. dissimilatory sulfate reduction in freshwater, the various associations between types of phototrophy and carbon fixation, and the density and diversity of glycoside hydrolases in freshwater microbes. We also investigated aspects of central metabolism such as hydrogen metabolism, oxidative phosphorylation, methylotrophy, and sugar degradation. Finally, by analyzing the dynamics over time in nitrogen fixation genes and Cyanobacteria genomes, we show that the potential for nitrogen fixation is linked to specific populations in Lake Mendota. This work represents an important step towards incorporating microbial data into ecosystem models and provides a better understanding of how microbes may participate in freshwater biogeochemical cycling., Competing Interests: The authors declare that they have no competing interests.

Known giant virus diversity is currently skewed towards viruses isolated from aquatic environments and cultivated in the laboratory. Here, we employ cultivation-independent metagenomics and mini-metagenomics on soils from the Harvard Forest, leading to the discovery of 16 novel giant viruses, chiefly recovered by mini-metagenomics. The candidate viruses greatly expand phylogenetic diversity of known giant viruses and either represented novel lineages or are affiliated with klosneuviruses, Cafeteria roenbergensis virus or tupanviruses. One assembled genome with a size of 2.4 Mb represents the largest currently known viral genome in the Mimiviridae, and others encode up to 80% orphan genes. In addition, we find more than 240 major capsid proteins encoded on unbinned metagenome fragments, further indicating that giant viruses are underexplored in soil ecosystems. The fact that most of these novel viruses evaded detection in bulk metagenomes suggests that mini-metagenomics could be a valuable approach to unearth viral giants.

Anaerobic ammonium oxidation (anammox) contributes substantially to ocean nitrogen loss, particularly in anoxic marine zones (AMZs). Ammonium is scarce in AMZs, raising the hypothesis that organic nitrogen compounds may be ammonium sources for anammox. Biochemical measurements suggest that the organic compounds urea and cyanate can support anammox in AMZs. However, it is unclear if anammox bacteria degrade these compounds to ammonium themselves, or rely on other organisms for this process. Genes for urea degradation have not been found in anammox bacteria, and genomic evidence for cyanate use for anammox is limited to a cyanase gene recovered from the sediment bacterium Candidatus Scalindua profunda. Here, analysis of Ca. Scalindua single amplified genomes from the Eastern Tropical North Pacific AMZ revealed genes for urea degradation and transport, as well as for cyanate degradation. Urease and cyanase genes were transcribed, along with anammox genes, in the AMZ core where anammox rates peaked. Homologs of these genes were also detected in meta-omic datasets from major AMZs in the Eastern Tropical South Pacific and Arabian Sea. These results suggest that anammox bacteria from different ocean regions can directly access organic nitrogen substrates. Future studies should assess if and under what environmental conditions these substrates contribute to the ammonium budget for anammox.

Background: 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., Results: From 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs., Conclusions: This population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

An enormous diversity of previously unknown bacteria and archaea has been discovered recently, yet their functional capacities and distributions in the terrestrial subsurface remain uncertain. Here, we continually sampled a CO 2 -driven geyser (Colorado Plateau, Utah, USA) over its 5-day eruption cycle to test the hypothesis that stratified, sandstone-hosted aquifers sampled over three phases of the eruption cycle have microbial communities that differ both in membership and function. Genome-resolved metagenomics, single-cell genomics and geochemical analyses confirmed this hypothesis and linked microorganisms to groundwater compositions from different depths. Autotrophic Candidatus "Altiarchaeum sp." and phylogenetically deep-branching nanoarchaea dominate the deepest groundwater. A nanoarchaeon with limited metabolic capacity is inferred to be a potential symbiont of the Ca. "Altiarchaeum". Candidate Phyla Radiation bacteria are also present in the deepest groundwater and they are relatively abundant in water from intermediate depths. During the recovery phase of the geyser, microaerophilic Fe- and S-oxidizers have high in situ genome replication rates. Autotrophic Sulfurimonas sustained by aerobic sulfide oxidation and with the capacity for N 2 fixation dominate the shallow aquifer. Overall, 104 different phylum-level lineages are present in water from these subsurface environments, with uncultivated archaea and bacteria partitioned to the deeper subsurface.

Virophages are small viruses that co-infect eukaryotic cells alongside giant viruses (Mimiviridae) and hijack their machinery to replicate. While two types of virophages have been isolated, their genomic diversity and ecology remain largely unknown. Here we use time series metagenomics to identify and study the dynamics of 25 uncultivated virophage populations, 17 of which represented by complete or near-complete genomes, in two North American freshwater lakes. Taxonomic analysis suggests that these freshwater virophages represent at least three new candidate genera. Ecologically, virophage populations are repeatedly detected over years and evolutionary stable, yet their distinct abundance profiles and gene content suggest that virophage genera occupy different ecological niches. Co-occurrence analyses reveal 11 virophages strongly associated with uncultivated Mimiviridae, and three associated with eukaryotes among the Dinophyceae, Rhizaria, Alveolata, and Cryptophyceae groups. Together, these findings significantly augment virophage databases, help refine virophage taxonomy, and establish baseline ecological hypotheses and tools to study virophages in nature.Virophages are recently-identified small viruses that infect larger viruses, yet their diversity and ecological roles are poorly understood. Here, Roux and colleagues present time series metagenomics data revealing new virophage genera and their putative ecological interactions in two freshwater lakes.

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia . Genome analysis revealed their potential role as (poly)saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called " Planctomycete -specific" cytochrome c -encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called " Planctomycete -specific" cytochrome c -encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia , while highlighting their potential role in freshwater carbon cycling.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

An explosion in the number of available genome sequences obtained through metagenomics and single-cell genomics has enabled a new view of the diversity of microbial life, yet we know surprisingly little about how microbes interact with each other or their environment. In fact, the majority of microbial species remain uncultivated, while our perception of their ecological niches is based on reconstruction of their metabolic potential. In this work, we demonstrate how the "seed set framework," which computes the set of compounds that an organism must acquire from its environment (E. Borenstein, M. Kupiec, M. W. Feldman, and E. Ruppin, Proc Natl Acad Sci U S A 105:14482-14487, 2008, https://doi.org/10.1073/pnas.0806162105), enables computational analysis of metabolic reconstructions while providing new insights into a microbe's metabolic capabilities, such as nutrient use and auxotrophies. We apply this framework to members of the ubiquitous freshwater actinobacterial lineage acI, confirming and extending previous experimental and genomic observations implying that acI bacteria are heterotrophs reliant on peptides and saccharides. We also present the first metatranscriptomic study of the acI lineage, revealing high expression of transport proteins and the light-harvesting protein actinorhodopsin. Putative transport proteins complement predictions of nutrients and essential metabolites while providing additional support of the hypothesis that members of the acI are photoheterotrophs. IMPORTANCE The metabolic activity of uncultivated microorganisms contributes to numerous ecosystem processes, ranging from nutrient cycling in the environment to influencing human health and disease. Advances in sequencing technology have enabled the assembly of genomes for these microorganisms, but our ability to generate reference genomes far outstrips our ability to analyze them. Common approaches to analyzing microbial metabolism require reconstructing the entirety of an organism's metabolic pathways or performing targeted searches for genes involved in a specific process. This paper presents a third approach, in which draft metabolic reconstructions are used to identify compounds through which an organism may interact with its environment. These compounds can then guide more-intensive metabolic reconstruction efforts and can also provide new hypotheses about the specific contributions that microbes make to ecosystem-scale metabolic processes.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world's largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth's most abundant organismal group., Competing Interests: The authors declare no competing financial interests in association with this study.

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

Multiple models describe the formation and evolution of distinct microbial phylogenetic groups. These evolutionary models make different predictions regarding how adaptive alleles spread through populations and how genetic diversity is maintained. Processes predicted by competing evolutionary models, for example, genome-wide selective sweeps vs gene-specific sweeps, could be captured in natural populations using time-series metagenomics if the approach were applied over a sufficiently long time frame. Direct observations of either process would help resolve how distinct microbial groups evolve. Here, from a 9-year metagenomic study of a freshwater lake (2005-2013), we explore changes in single-nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in 30 bacterial populations. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied by >1000-fold among populations. SNP allele frequencies also changed dramatically over time within some populations. Interestingly, nearly all SNP variants were slowly purged over several years from one population of green sulfur bacteria, while at the same time multiple genes either swept through or were lost from this population. These patterns were consistent with a genome-wide selective sweep in progress, a process predicted by the 'ecotype model' of speciation but not previously observed in nature. In contrast, other populations contained large, SNP-free genomic regions that appear to have swept independently through the populations prior to the study without purging diversity elsewhere in the genome. Evidence for both genome-wide and gene-specific sweeps suggests that different models of bacterial speciation may apply to different populations coexisting in the same environment.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active 'orphan' MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

Extensive genomic diversity within coexisting members of a microbial species has been revealed through selected cultured isolates and metagenomic assemblies. Yet, the cell-by-cell genomic composition of wild uncultured populations of co-occurring cells is largely unknown. In this work, we applied large-scale single-cell genomics to study populations of the globally abundant marine cyanobacterium Prochlorococcus. We show that they are composed of hundreds of subpopulations with distinct "genomic backbones," each backbone consisting of a different set of core gene alleles linked to a small distinctive set of flexible genes. These subpopulations are estimated to have diverged at least a few million years ago, suggesting ancient, stable niche partitioning. Such a large set of coexisting subpopulations may be a general feature of free-living bacterial species with huge populations in highly mixed habitats.