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Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we optimized a microarray single-cell sequencing (MASC-seq) technology that generates microscope images of cells in parallel with capturing their transcriptomes. We tested the method on three species representing important plankton groups with different cell structures, the ciliateTetrahymena thermophila, the diatomPhaeodactylum tricornutumand the dinoflagellateHeterocapsasp.. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating “microbial dark matter”, although further optimizations are necessary to increase the signal-to-noise ratio.

Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—is moderated by complex interactions between trees and soil microorganisms. However, existing methods limit our ability to predict how changes in environmental conditions will alter these associations and the essential ecosystem services they provide. To address this, we developed a metatranscriptomic approach to analyze the impact of nutrient enrichment on Norway spruce fine roots and the community structure, function, and tree–microbe coordination of over 350 root-associated fungal species. In response to altered nutrient status, host trees redefined their relationship with the fungal community by reducing sugar efflux carriers and enhancing defense processes. This resulted in a profound restructuring of the fungal community and a collapse in functional coordination between the tree and the dominant Basidiomycete species, and an increase in functional coordination with versatile Ascomycete species. As such, there was a functional shift in community dominance from Basidiomycetes species, with important roles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have melanized cell walls that are highly resistant to degradation. These changes were accompanied by prominent shifts in transcriptional coordination between over 60 predicted fungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanistic insight into the complex molecular dialogue coordinating host trees and their fungal partners. The host–microbe dynamics captured by this study functionally inform how these complex and sensitive biological relationships may mediate the carbon storage potential of boreal soils under changing nutrient conditions.

Microbial communities are major players in carbon and nitrogen cycling globally and are of particular importance for plant communities in the nutrient poor soils of boreal forests. Especially relevant are the fungal communities in the soil that interact with the plants in multiple ways, indirectly through their pivotal role in the breakdown of organic matter and, more directly, through mycorrhizal symbiosis with plant roots. Large-scale disturbances of these complex microbial communities can lead to shifts in soil carbon storage with unknown and global-scale long-term consequences. To understand the dynamics of these communities and their relationship to associated plants in response to climate change and anthropogenic influence, we need a better understanding of how modern “omics” methods can help us to understand compositional and functional shifts of these microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from environmental samples. In contrast, currently phylogenetic marker gene amplicon sequencing data is generally used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in RNA-Seq transcriptomic data from matched samples. Here we describe fungal communities using both RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from mature stands of the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient deficient) and nutrient enriched plots at the Flakaliden forest research site in boreal northern Sweden. We created an assembly-based, reproducible and hardware agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. We show that the community structure indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying limitations imposed by current database coverage. Furthermore, we show examples to demonstrate how metatranscriptomics data additionally provides biologically informative functional insight at the community and individual species level. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response and effect both between host plants and their associated microbial communities, and among the members of microbial communities in environmental samples in general.

The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus Cortinarius. IMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.

Extremely oligotrophic deep groundwaters host organisms attuned to the low-end of the bioenergetics spectrum. While all domains of life along with viruses are active in this habitat, the evolutionary and ecological constraints on colonization and niche shifts and their consequences for the microbiome convergence are unknown. Here we provide a comparative genome-resolved analysis of the prokaryotic community in disconnected fracture fluids of the Fennoscandian Shield. The data show that the oligotrophic deep groundwaters flowing in similar lithologies offer fixed niches that are occupied by a common deep groundwater core microbiome. Based on this high resolution “multi-omics” enabled understanding of the underlying mechanisms via functional expression analysis, we conclude that deep groundwater ecosystems foster highly diverse, yet cooperative microbial communities adapted to this setting. The fitness of primary energy producers is increased by ecological traits such as aggregate or biofilm formation. This also facilitates reciprocal promiscuous partnerships with diverse and prevalent epi-bionts, alleviating the “tragedy of common goods”. Hence, instead of a lifestyle where microbes predominantly invest in functions related to maintenance and survival, an episodic and cooperative lifestyle ensures the subsistence of the deep groundwater microbiome. We suggest the name “halt and catch fire” for this way of life.

The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract No. DE-AC02-05CH11231. The Swedish Research Council (contracts 2018-04311, 2017-04422, and 2014-4398) and The Swedish Nuclear Fuel and Waste Management Company (SKB) supported the study. M.D. thanks the Crafoord Foundation (contracts 20180599 and 20130557), the Nova Center for University Studies, Research and Development, and Familjen Hellmans Stiftelse for financial support. M.D. and D.S. thank the Carl Tryggers Foundation (grant KF16: 18) for financial support. S.B. and M.M. acknowledge financial support from the Swedish Research Council and Science for Life Laboratory. High-throughput sequencing was also carried out at the National Genomics Infrastructure hosted by the Science for Life Laboratory. Bioinformatics analyses were carried out utilizing the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) at Uppsala University (projects b2013127, SNIC 2019/3-22, and SNIC 2020/5-19) with support from a SciLifeLab-WABI bioinformatics grant. We would also like to thank Mats Astrom for his comments on the Aspo HRL lithology. JS is financially supported by the Knut and Alice Wallenberg Foundation as part of the National Bioinformatics Infrastructure Sweden at SciLifeLab., While oligotrophic deep groundwaters host active microbes attuned to the low-end of the bioenergetics spectrum, the ecological constraints on microbial niches in these ecosystems and their consequences for microbiome convergence are unknown. Here, we provide a genome-resolved, integrated omics analysis comparing archaeal and bacterial communities in disconnected fracture fluids of the Fennoscandian Shield in Europe. Leveraging a dataset that combines metagenomes, single cell genomes, and metatranscriptomes, we show that groundwaters flowing in similar lithologies offer fixed niches that are occupied by a common core microbiome. Functional expression analysis highlights that these deep groundwater ecosystems foster diverse, yet cooperative communities adapted to this setting. We suggest that these communities stimulate cooperation by expression of functions related to ecological traits, such as aggregate or biofilm formation, while alleviating the burden on microorganisms producing compounds or functions that provide a collective benefit by facilitating reciprocal promiscuous metabolic partnerships with other members of the community. We hypothesize that an episodic lifestyle enabled by reversible bacteriostatic functions ensures the subsistence of the oligotrophic deep groundwater microbiome., United States Department of Energy (DOE) DE-AC02-05CH11231, Swedish Research Council, European Commission 2018-04311 2017-04422 2014-4398, Swedish Nuclear Fuel and Waste Management Company (SKB), Crafoord Foundation 20180599 20130557, Nova Center for University Studies, Research and Development, Familjen Hellmans Stiftelse, Carl Tryggers Foundation KF16: 18, Swedish Research Council European Commission, Science for Life Laboratory, Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) at Uppsala University b2013127 SNIC 2019/3-22 SNIC 2020/5-19, SciLifeLab-WABI bioinformatics grant, Knut and Alice Wallenberg Foundation as part of the National Bioinformatics Infrastructure Sweden at SciLifeLab

Segmented filamentous bacteria (SFB) are unique immune modulatory bacteria colonizing the small intestine of a variety of animals in a host-specific manner. SFB exhibit filamentous growth and attach to the host’s intestinal epithelium, offering a physical route of interaction. SFB affect functions of the host immune system, among them IgA production and T-cell maturation. Until now, no human-specific SFB genome has been reported. Here, we report the metagenomic reconstruction of an SFB genome from a human ileostomy sample. Phylogenomic analysis clusters the genome with SFB genomes from mouse, rat and turkey, but the genome is genetically distinct, displaying 65–71% average amino acid identity to the others. By screening human faecal metagenomic datasets, we identified individuals carrying sequences identical to the new SFB genome. We thus conclude that a unique SFB variant exists in humans and foresee a renewed interest in the elucidation of SFB functionality in this environment., Hans Jonsson et al. report the metagenomic reconstruction of the genome of a potentially immune modulatory segmented filamentous bacteria (SFB) from a human ileostomy sample. They demonstrate that the genome clusters closely with SFB genomes from other species. They also detect the unique SFB variant in human faecal metagenomics datasets.

SummarySegmented filamentous bacteria (SFB) colonize the small intestine of a variety of animals in a host-specific manner. SFB are physically attached to the host’s intestinal epithelium and affect several functions related to the immune system, among them IgA production and T-cell maturation. Until now, no human-specific SFB genome had been described. Here, we report the metagenomic reconstruction of an SFB genome from a human ileostomy sample. Phylogenomic analysis clusters the genome with the SFB genomes from mouse, rat and turkey, but the genome is genetically distinct, displaying 65-71% average amino acid identity to the other genomes, and is tentatively unique for the human small intestine. By screening human faecal metagenomic datasets, we identified individuals carrying sequences identical to the new SFB-genome. We thus conclude that a unique SFB variant exists in humans and we foresee a renewed interest in the elucidation of SFB functionality in this environment.

The Baltic Sea is one of the world’s largest brackish water bodies and is characterised by pronounced physicochemical gradients where microbes are the main biogeochemical catalysts. Meta-omic methods provide rich information on the composition of, and activities within, microbial ecosystems, but are computationally heavy to perform. We here present the Baltic Sea Reference Metagenome (BARM), complete with annotated genes to facilitate further studies with much less computational effort. The assembly is constructed using 2.6 billion metagenomic reads from 81 water samples, spanning both spatial and temporal dimensions, and contains 6.8 million genes that have been annotated for function and taxonomy. The assembly is useful as a reference, facilitating taxonomic and functional annotation of additional samples by simply mapping their reads against the assembly. This capability is demonstrated by the successful mapping and annotation of 24 external samples. In addition, we present a public web interface, BalticMicrobeDB, for interactive exploratory analysis of the dataset.

Cyanobacteria are important phytoplankton in the Baltic Sea, an estuarine-like environment with pronounced north to south gradients in salinity and nutrient concentrations. Here, we present a metagenomic and -transcriptomic survey, with subsequent analyses targeting the genetic identity, phylogenetic diversity, and spatial distribution of Baltic Sea cyanobacteria. The cyanobacterial community constituted close to 12% of the microbial population sampled during a pre-bloom period (June-July 2009). The community was dominated by unicellular picocyanobacteria, specifically a few highly abundant taxa (Synechococcus and Cyanobium) with a long tail of low abundance representatives, and local peaks of bloom-forming heterocystous taxa. Cyanobacteria in the Baltic Sea differed genetically from those in adjacent limnic and marine waters as well as from cultivated and sequenced picocyanobacterial strains. Diversity peaked at brackish salinities 3.5-16 psu, with low N:P ratios. A shift in community composition from brackish to marine strains was accompanied by a change in the repertoire and expression of genes involved in salt acclimation. Overall, the pre-bloom cyanobacterial population was more genetically diverse, widespread and abundant than previously documented, with unicellular picocyanobacteria being the most abundant clade along the entire Baltic Sea salinity gradient.

Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response, and programmed cell death (PCD) in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to ~4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (>0.8 μm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e., sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.