The National Microbiome Data Collaborative Data Portal: an integrated multi-omics microbiome data resource
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Seagrasses are marine flowering plants that provide critical ecosystem services in coastal environments worldwide. Marine fungi are often overlooked in microbiome and seagrass studies, despite terrestrial fungi having critical functional roles as decomposers, pathogens, or endophytes in global ecosystems. Here, we characterize the distribution of fungi associated with the seagrass Zostera marina, using leaves, roots, and rhizosphere sediment from 16 locations across its full biogeographic range. Using high-throughput sequencing of the ribosomal internal transcribed spacer (ITS) region and 18S rRNA gene, we first measured fungal community composition and diversity. We then tested hypotheses of neutral community assembly theory and the degree to which deviations suggested that amplicon sequence variants (ASVs) were plant selected or dispersal limited. Finally, we identified a core mycobiome and investigated the global distribution of differentially abundant ASVs. We found that the fungal community is significantly different between sites and that the leaf mycobiome follows a weak but significant pattern of distance decay in the Pacific Ocean. Generally, there was evidence for both deterministic and stochastic factors contributing to community assembly of the mycobiome, with most taxa assembling through stochastic processes. The Z. marina core leaf and root mycobiomes were dominated by unclassified Sordariomycetes spp., unclassified Chytridiomycota lineages (including Lobulomycetaceae spp.), unclassified Capnodiales spp., and Saccharomyces sp. It is clear from the many unclassified fungal ASVs and fungal functional guilds that knowledge of marine fungi is still rudimentary. Further studies characterizing seagrass-associated fungi are needed to understand the roles of these microorganisms generally and when associated with seagrasses. Fungi have important functional roles when associated with land plants, yet very little is known about the roles of fungi associated with marine plants, like seagrasses. In this study, we report the results of a global effort to characterize the fungi associated with the seagrass Zostera marina across its full biogeographic range. Although we defined a putative global core fungal community, it is apparent from the many fungal sequences and predicted functional guilds that had no matches to existing databases that general knowledge of seagrass-associated fungi and marine fungi is lacking. This work serves as an important foundational step toward future work investigating the functional ramifications of fungi in the marine ecosystem.
Protists, in particular bacterivores, are essential players in the rhizosphere; thus, how their interactions with bacteria and fungi affect plant productivity and soil nutrient cycles warrants more attention. Using next-generation sequencing of the 18 S rRNA gene, we investigated the distribution of two major protistan phyla, Cercozoa and Endomyxa, across four seasons, and four soil compartments - rhizosphere, root, soil and litter. The sampling was replicated in two forests in Norway and the Czech Republic, in order to test our results across biogeographic scales. Compartment had a major influence in shaping protistan communities, over and above spatial distance and seasonal variation. Protistan diversity was highest in the bulk soil while lowest in the roots, suggesting that the plants select for restricted assemblages of protists. Accordingly, only the root compartment harboured a subset of the bulk soil protistan diversity. In addition, protistan communities showed markedly different distributions according to their feeding modes, with opposite patterns for bacterivores versus omnivores and eukaryvores. The small bacterivorous flagellates (mostly Glissomonadida) were more abundant in roots, while the larger amoeboid eukaryvores (e.g. some of the Cryomonadida and vampyrellids) dominated in soil and in the rhizosphere, and the omnivores (e.g. Euglyphida and part of the Cercomonadida), also large and mostly amoeboid, were more abundant in litter. The current view of the soil microbiome is mostly focused on bacteria and fungi: this detailed study on the community distribution of protists according to their feeding modes reveals the essential role they play in each of the soil compartments, an essential precondition for a detailed understanding of the soil food web and nutrient cycling in forest.
Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions.
Abstract Environmental and host-associated microbiomes are typically diverse assemblages of organisms performing myriad activities and engaging in a network of interactions that play out in spatially structured contexts. As the sum of these activities and interactions give rise to overall microbiome function, with important consequences for environmental processes and human health, elucidating specific microbial activities within complex communities is a pressing challenge. Single-cell stable isotope probing (SC-SIP) encompasses multiple techniques that typically utilize Raman microspectroscopy or nanoscale secondary ion mass spectrometry (NanoSIMS) to enable spatially resolved tracking of isotope tracers in cells, cellular components, and metabolites. SC-SIP techniques are uniquely suited for illuminating single-cell activities in microbial communities and for testing hypotheses about cellular functions generated for example from meta-omics datasets. Here, we illustrate the insights enabled by SC-SIP techniques by reviewing selected applications in microbiology and offer a perspective on their potential for future research.
Subsurface environments host diverse microorganisms in fluid-filled fractures; however, little is known about how geological and hydrological processes shape the subterranean biosphere. Here, we sampled three flowing boreholes weekly for 10 mo in a 1478-m-deep fractured rock aquifer to study the role of fracture activity (defined as seismically or aseismically induced fracture aperture change) and advection on fluid-associated microbial community composition. We found that despite a largely stable deep-subsurface fluid microbiome, drastic community-level shifts occurred after events signifying physical changes in the permeable fracture network. The community-level shifts include the emergence of microbial families from undetected to over 50% relative abundance, as well as the replacement of the community in one borehole by the earlier community from a different borehole. Null-model analysis indicates that the observed spatial and temporal community turnover was primarily driven by stochastic processes (as opposed to deterministic processes). We, therefore, conclude that the observed community-level shifts resulted from the physical transport of distinct microbial communities from other fracture(s) that outpaced environmental selection. Given that geological activity is a major cause of fracture activity and that geological activity is ubiquitous across space and time on Earth, our findings suggest that advection induced by geological activity is a general mechanism shaping the microbial biogeography and diversity in deep-subsurface habitats across the globe.
Summary Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography–tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual‐identified exometabolites. Lastly, we compared responses of a P. tricornutum ‐adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4‐hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4‐hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.
Plants can influence soil microbes through resource acquisition and interference competition, with consequences for ecosystem function such as nitrification. However, how plants alter soil conditions to influence nitrifiers and nitrification rates remains poorly understood, especially in the subsoil. Here, coupling the 15N isotopic pool dilution technique, high throughput sequencing and in situ soil O2 monitoring, we investigated how a deep-rooted perennial grass, miscanthus, versus an adjacent shallow-rooted turfgrass reference shapes nitrifier assembly and function along 1 m soil profiles. In topsoil, the suppression of ammonia (NH3) oxidizing archaea (AOA) and gross nitrification rates in miscanthus relative to the reference likely resulted from nitrifiers being outcompeted by plant roots and heterotrophic bacteria for ammonium (NH4+). The stronger tripartite competition under miscanthus may have been caused in part by the lower soil organic matter (SOM) content, which supported lower gross nitrogen (N) mineralization, the major soil process that produces NH4+. In contrast, below 10 cm soil depth, significantly greater gross nitrification rates were observed in miscanthus compared to the reference. This was likely driven by the significantly lower oxygen (O2) in miscanthus than reference subsoil, which selected against aerobic heterotrophic bacteria but in favor of AOA. Overall, we found that plants can regulate AOA community structure and function through different mechanisms in topsoil and subsoil, with suppression of nitrification in topsoil and enhancement of nitrification in subsoil.
Abstract Background Despite their widespread distribution and ecological importance, protists remain one of the least understood components of the soil and rhizosphere microbiome. Knowledge of the roles that protists play in stimulating organic matter decomposition and shaping microbiome dynamics continues to grow, but there remains a need to understand the extent to which biological and environmental factors mediate protist community assembly and dynamics. We hypothesize that protists communities are filtered by the influence of plants on their rhizosphere biological and physicochemical environment, resulting in patterns of protist diversity and composition that mirror previously observed diversity and successional dynamics in rhizosphere bacterial communities. Results We analyzed protist communities associated with the rhizosphere and bulk soil of switchgrass (SG) plants ( Panicum virgatum ) at different phenological stages, grown in two marginal soils as part of a large-scale field experiment. Our results reveal that the diversity of protists is lower in rhizosphere than bulk soils, and that temporal variations depend on soil properties but are less pronounced in rhizosphere soil. Patterns of significantly prevalent protists groups in the rhizosphere suggest that most protists play varied ecological roles across plant growth stages and that some plant pathogenic protists and protists with omnivorous diets reoccur over time in the rhizosphere. We found that protist co-occurrence network dynamics are more complex in the rhizosphere compared to bulk soil. A phylogenetic bin-based null model analysis showed that protists’ community assembly in our study sites is mainly controlled by homogenous selection and dispersal limitation, with stronger selection in rhizosphere than bulk soil as SG grew and senesced. Conclusions We demonstrate that environmental filtering is a dominant determinant of overall protist community properties and that at the rhizosphere level, plant control on the physical and biological environment is a critical driver of protist community composition and dynamics. Since protists are key contributors to plant nutrient availability and bacterial community composition and abundance, mapping and understanding their patterns in rhizosphere soil is foundational to understanding the ecology of the root-microbe-soil system.
Rhizosphere microbiomes and root exudates play a pivotal biochemical role in helping to catalyze chitin catabolism. Chitin is a recalcitrant and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, the biochemical mechanisms controlling chitin fate in the rhizosphere are elusive and poorly understood. To enable spatial mapping of chitinase activity in the rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a high fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer of chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offer non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. Furthermore, we observed increased chitinase activity near plant roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase activity with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can help enlighten efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.
We present observations from a laboratory-controlled study on the impacts of extreme wetting and drying on a wetland soil microbiome. Our approach was to experimentally challenge the soil microbiome to understand impacts on anaerobic carbon cycling processes as the system transitions from dryness to saturation and vice-versa. Specifically, we tested for impacts on stress responses related to shifts from wet to drought conditions. We used a combination of high-resolution data for small organic chemical compounds (metabolites) and biological (community structure based on 16S rRNA gene sequencing) features. Using a robust correlation-independent data approach, we further tested the predictive power of soil metabolites for the presence or absence of taxa. Here, we demonstrate that taking an untargeted, multidimensional data approach to the interpretation of metabolomics has the potential to indicate the causative pathways selecting for the observed bacterial community structure in soils.
Despite a growing understanding of how environmental composition affects microbial communities, it remains difficult to apply this knowledge to the rational design of synthetic multispecies consortia. This is because natural microbial communities can harbour thousands of different organisms and environmental substrates, making up a vast combinatorial space that precludes exhaustive experimental testing and computational prediction. Here, we present a method based on the combination of machine learning and metabolic modelling that selects optimal environmental compositions to produce target community phenotypes. In this framework, dynamic flux balance analysis is used to model the growth of a community in candidate environments. A genetic algorithm is then used to evaluate the behaviour of the community relative to a target phenotype, and subsequently adjust the environment to allow the organisms to approach this target. We apply this iterative process to thousands of in silico communities of varying sizes, showing how it can rapidly identify environments that yield desired taxonomic compositions and patterns of metabolic exchange. Moreover, this combination of approaches produces testable predictions for the assembly of experimental microbial communities with specific properties and can facilitate rational environmental design processes for complex microbiomes.
Background - Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. Results - We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. Conclusions - Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.
Advancing the bioeconomy requires the development of large-scale microbial bioprocesses capable of converting waste carbon streams into biofuels, biochemicals, and biomaterials at industrially relevant scales. While biomanufacturing has been successfully demonstrated at the laboratory scale for a wide range of chemicals, only a few have reached industrial-scale production. This is partly due to the inherent complexity of microbial systems, which rely on living cells with intricate metabolic pathways that are highly sensitive to environmental changes, making large-scale production difficult to optimize and predict. As a result, scaling-up bioprocesses remains a high-stakes challenge that requires deeper exploration. This involves integrating feedstock and microbial selection, upstream and downstream processes, and computational modelling, among other research efforts. Bulk and specialty chemicals derived from biological processes also face competition from fossil-based production routes, which have been refined through decades of technological advancements. While biologically derived molecules may offer more environmentally friendly production pathways than traditional chemical manufacturing, their widespread adoption depends on achieving cost parity-or superiority-relative to fossil-based methods. This emphasizes the importance of holistic research, including techno-economic analyses and life cycle assessments, to ensure both economic viability and environmental sustainability. This editorial and special issue explores state-of-the-art strategies for converting waste carbon sources into valuable products. It discusses how enzymes, single microbes (e.g., extremophiles), and microbiomes (e.g., through division of labor) can be integrated with upstream and downstream process innovations-such as consolidated bioprocessing and in situ product recovery-to improve the efficiency and scalability of biomanufacturing. The editorial further highlights the role of computational modelling in understanding, predicting, and controlling bioprocess performance across scales, and concludes by emphasizing the importance of techno-economic modelling to identify technologies that can move to market.
We evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4–V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1–V3(1) and (2), and V3–V4 and V3–V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.
The eastern oysters serve as important keystone species in the United States, especially in the Gulf of Mexico estuarine waters, and at the same time, provide unparalleled economic, ecological, environmental, and cultural services. One ecosystem service that has garnered recent attention is the ability of oysters to sequester impurities and nutrients, such as nitrogen (N), from the estuarine water that feeds them, via their exceptional filtration mechanism coupled with microbially-mediated denitrification processes. It is the oyster-associated microbiomes that essentially provide these myriads of ecological functions, yet not much is known on these microbiota at the genomic scale, especially from warm temperate and tropical water habitats. Among the suite of bacterial genera that appear to interplay with the oyster host species, pseudomonads deserve further assessment because of their immense metabolic and ecological potential. To obtain a comprehensive understanding on this aspect, we previously reported on the isolation and preliminary genomic characterization of three Pseudomonas species isolated from minced oyster tissue (P. alcaligenes strain OT69); oyster mantle fluid (P. stutzeri strain MF28) and the water collected from top of the oyster reef (P. aeruginosa strain WC55), respectively. In this comparative genomic analysis study conducted on these three targeted pseudomonads, native to the eastern oyster and its surrounding environment, provided further insights into their unique functional traits, conserved gene pools between the selected pseudomonads, as well as genes that render unique characteristics in context to metabolic traits recruited during their evolutionary history via horizontal gene transfer events as well as phage-mediated incorporation of genes. Moreover, the strains also supported extensively developed resistomes, which suggests that environmental microorganisms native to relatively pristine environments, such as Apalachicola Bay, Florida, have also recruited an arsenal of antibiotic resistant gene determinants, thus posing an emerging public health concern.
Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.
Age and gender have been recognized as two pivotal covariates affecting the composition of the gut microbiota. However, their mediated variations in microbiota seem to be inconsistent across different countries and races. In this study, 613 individuals, whom we referred to as the “healthy” population, were selected from 1,018 volunteers through rigorous selection using 16S rRNA sequencing. Three enterotypes were identified, namely, Escherichia–Shigella , mixture ( Bacteroides and Faecalibacterium ), and Prevotella . Moreover, 11 covariates that explain the differences in microbiota were determined, with age being the predominant factor. Furthermore, age-related differences in alpha diversity, beta diversity, and core genera were observed in our cohort. Remarkably, after adjusting for 10 covariates other than age, abundant genera that differed between age groups were demonstrated. In contrast, minimal differences in alpha diversity, beta diversity, and differentially abundant genera were observed between male and female individuals. Furthermore, we also demonstrated the age trajectories of several well-known beneficial genera, lipopolysaccharide (LPS)-producing genera, and short-chain fatty acids (SCFAs)-producing genera. Overall, our study further elucidated the effects mediated by age and gender on microbiota differences, which are of significant importance for a comprehensive understanding of the gut microbiome spectrum in healthy individuals.