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Root-colonizing fungi can form mycorrhizal or endophytic associations with plant roots, the type of association depending on the host. We investigated the differences and similarities of the fungal communities of three boreal ericoid plants and one coniferous tree, and identified the community structure of fungi utilizing photosynthates from the plants studied. The fungal communities of roots and soils of Vaccinium myrtillus, Vaccinium vitis-idaea, Calluna vulgaris and Pinus sylvestris were studied in an 18-month-long experiment where the plants were grown individually in natural substrate. Photosynthates utilizing fungi were detected with DNA stable-isotope probing using 13CO2 (13C-DNA-SIP). The results indicated that the plants studied provide different ecological niches preferred by different fungal species. Those fungi which dominated the community in washed roots had also the highest 13C-uptake. In addition, a common root endophyte without confirmed mycorrhizal status also obtained 13C from all the plants, indicating close plant-association of this fungal species. We detect several fungal species inhabiting the roots of both ericoid mycorrhizal and ectomycorrhizal plants. Our results highlight that the ecological role of co-occurrence of fungi with different life styles (e.g. mycorrhizal or endophytic) in plant root systems should be further investigated.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

The aim of this study was to gain understanding of the carbon flow from the roots of a genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar and its parental isoline to the soil fungal community using stable isotope probing (SIP). The microbes receiving 13C from the plant were assessed through RNA/phospholipid fatty acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism (T-RFLP). Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate of carbon allocation in the GM variety differed from that in its parental variety, thereby affecting soil fungal communities. We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates flowing from the root into the rhizosphere, that there are large differences in utilization of root-derived compounds at a lower phylogenetic level within investigated fungal phyla, and that active communities in the rhizosphere differ between the GM plant and its parental cultivar through effects of differential carbon flow from the plant.

Understanding how population‐level dynamics contribute to ecosystem‐level processes is a primary focus of ecological research and has led to important breakthroughs in the ecology of macroscopic organisms. However, the inability to measure population‐specific rates, such as growth, for microbial taxa within natural assemblages has limited ecologists’ understanding of how microbial populations interact to regulate ecosystem processes. Here, we use isotope incorporation within DNA molecules to model taxon‐specific population growth in the presence of 18O‐labeled water. By applying this model to phylogenetic marker sequencing data collected from stable‐isotope probing studies, we estimate rates of growth, mortality, and turnover for individual microbial populations within soil assemblages. When summed across the entire bacterial community, our taxon‐specific estimates are within the range of other whole‐assemblage measurements of bacterial turnover. Because it can be applied to environmental samples, the approach we present is broadly applicable to measuring population growth, mortality, and associated biogeochemical process rates of microbial taxa for a wide range of ecosystems and can help reveal how individual microbial populations drive biogeochemical fluxes.

Plant roots harbor a large diversity of microorganisms that have an essential role in ecosystem functioning. To better understand the level of intimacy of root-inhabiting microbes such as arbuscular mycorrhizal fungi and bacteria, we provided ¹³CO₂ to plants at atmospheric concentration during a 5-h pulse. We expected microbes dependent on a carbon flux from their host plant to become rapidly labeled. We showed that a wide variety of microbes occurred in roots, mostly previously unknown. Strikingly, the greatest part of this unsuspected diversity corresponded to active primary consumers. We found 17 bacterial phylotypes co-occurring within roots of a single plant, including five potentially new phylotypes. Fourteen phylotypes were heavily labeled with the ¹³C. Eight were phylogenetically close to Burkholderiales, which encompass known symbionts; the others were potentially new bacterial root symbionts. By analyzing unlabeled and ¹³C-enriched RNAs, we demonstrated differential activity in C consumption among these root-inhabiting microbes. Arbuscular mycorrhizal fungal RNAs were heavily labeled, confirming the high carbon flux from the plant to the fungal compartment, but some of the fungi present appeared to be much more active than others. The results presented here reveal the possibility of uncharacterized root symbioses.

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. 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.

Rice paddy fields are sustainable agricultural systems as soil microorganisms help maintain nitrogen fertility through generating ammonium. In these soils, dissimilatory nitrate reduction to ammonium (DNRA), nitrogen fixation, and denitrification are closely linked. DNRA and denitrification share the same initial steps and nitrogen gas, the end product of denitrification, can serve as a substrate for nitrogen fixation. However, the microorganisms responsible for these three reductive nitrogen transformations, particularly those focused on ammonium generation, have not been comprehensively characterized. In this study, we used stable isotope probing with 15NO3−, 15N2O, and 15N2, combined with 16S rRNA high-throughput sequencing and metatranscriptomics, to identify ammonium-generating microbial consortia in paddy soils. Our results revealed that several bacterial families actively contribute to ammonium generation under different nitrogen substrate conditions. Specifically, Geobacteraceae (N2O and +N2), Bacillaceae (+NO3− and +N2), Rhodocyclaceae (+N2O and +N2), Anaeromyxobacteraceae (+NO3− and +N2O), and Clostridiaceae (+NO3− and +N2) were involved. Many of these bacteria participate in key ecological processes typical of paddy environments, including iron or sulfate reduction and rice straw decomposition. This study revealed the ammonium-generating microbial consortia in paddy soil that contain several key bacterial drivers of multiple reductive nitrogen transformations and suggested their diverse functions in paddy soil metabolism.

Stable isotope probing of prokaryotic DNA was used to determine active prokaryotes using ¹³C-labelled substrates (glucose, acetate, CO₂) in sediment slurries from different biogeochemical zones of the Severn Estuary, UK. Multiple, low concentrations (5 x 100 μM) of ¹³C-substrate additions and short-term incubations (7 days) were used to minimize changes in the prokaryotic community, while achieving significant ¹³C-incorporation. Analysis demonstrated clear metabolic activity within all slurries, although neither the net sulphate removal nor CH₄ production occurred in the anaerobic sulphate reduction and methanogenesis zone slurries. Some similarities occurred in the prokaryotic populations that developed in different sediment slurries, particularly in the aerobic and dysaerobic zone slurries with ¹³C-glucose, which were dominated by Gammaproteobacteria and Marine Group 1 Archaea, whereas both anaerobic sediment slurries incubated with ¹³C-acetate showed incorporation into Epsilonproteobacteria and other bacteria, with the sulphate reduction zone slurry also showing ¹³C-acetate utilization by Miscellaneous Crenarchaeotic Group Archaea. The lower potential energy methanogenesis zone slurries were the only conditions where no ¹³C-incorporation into Archaea occurred, despite Bacteria being labelled; this was surprising because Archaea have been suggested to be adapted to low-energy conditions. Overall, our results highlight that uncultured prokaryotes play important ecological roles in tidal sediments of the Severn Estuary, providing new metabolic information for novel groups of Archaea and suggesting broader metabolisms for largely uncultivated Bacteria.

The structure and function of a microbial community capable of biodegrading methyl-tert-butyl ether (MTBE) was characterized using compound-specific stable isotope analysis (CSIA), clone libraries and stable isotope probing of proteins (Protein-SIP). The enrichment culture (US3-M), which originated from a gasoline-impacted site in the United States, has been enriched on MTBE as the sole carbon source. The slope of isotopic enrichment factors (εC of -2.29±0.03[per thousand]; εH of -58±6[per thousand]) for carbon and hydrogen discrimination (Δδ²H/Δδ¹³C) was on average equal to Λ=24±2, a value closely related to the reaction mechanism of MTBE degradation in Methylibium petroleiphilum PM1. 16S rRNA gene libraries revealed sequences belonging to M. petroleiphilum PM1, Hydrogenophaga sp., Thiothrix unzii, Rhodobacter sp., Nocardiodes sp. and different Sphingomonadaceae bacteria. Protein-SIP analysis of the culture grown on ¹³C-MTBE as the only carbon source revealed that proteins related to members of the Comamonadaceae family, such as Delftia acidovorans, Acidovorax sp. or Comamonas sp., were not ¹³C-enriched, whereas proteins related to M. petroleiphilum PM1 showed an average incorporation of 94.5 atom%¹³C. These results indicate a key role for this species in the degradation of MTBE within the US3-M consortia. The combination of CSIA, molecular biology and Protein-SIP facilitated the analysis of an MTBE-degrading mixed culture from a functional and phylogenetic point of view.