Dataset Information

Quantitative microbial ecology through stable isotope probing.

ABSTRACT: Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in (18)O and (13)C composition after exposure to [(18)O]water or [(13)C]glucose. The addition of glucose increased the assimilation of (18)O into DNA from [(18)O]water. However, the increase in (18)O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

SUBMITTER: Hungate BA

PROVIDER: S-EPMC4592864 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Quantitative microbial ecology through stable isotope probing.

Hungate Bruce A BA Mau Rebecca L RL Schwartz Egbert E Caporaso J Gregory JG Dijkstra Paul P van Gestel Natasja N Koch Benjamin J BJ Liu Cindy M CM McHugh Theresa A TA Marks Jane C JC Morrissey Ember M EM Price Lance B LB

Applied and environmental microbiology 20150821 21

Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic c ...[more]

PMID: 26296731

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Project description:Benthic environments harbor highly diverse and complex microbial communities that control carbon fluxes, but the role of specific uncultivated microbial groups in organic matter turnover is poorly understood. In this study, quantitative DNA stable isotope probing (DNA-qSIP) was used for the first time to link uncultivated populations of bacteria and archaea to carbon turnover in lacustrine surface sediments. After 1-week incubations in the dark with [13C]bicarbonate, DNA-qSIP showed that ammonia-oxidizing archaea (AOA) were the dominant active chemolithoautotrophs involved in the production of new organic matter. Natural 13C-labeled organic matter was then obtained by incubating sediments in the dark for 2.5 months with [13C]bicarbonate, followed by extraction and concentration of high-molecular-weight (HMW) (>50-kDa) organic matter. qSIP showed that the labeled organic matter was turned over within 1 week by 823 microbial populations (operational taxonomic units [OTUs]) affiliated primarily with heterotrophic Proteobacteria, Chloroflexi, Verrucomicrobia, and Bacteroidetes However, several OTUs affiliated with the candidate microbial taxa Latescibacteria, Omnitrophica, Aminicentantes, Cloacimonates, AC1, Bathyarchaeota, and Woesearchaeota, groups known only from genomic signatures, also contributed to biomass turnover. Of these 823 labeled OTUs, 52% (primarily affiliated with Proteobacteria) also became labeled in 1-week incubations with [13C]bicarbonate, indicating that they turned over carbon faster than OTUs that were labeled only in incubations with 13C-labeled HMW organic matter. These taxa consisted primarily of uncultivated populations within the Firmicutes, Bacteroidetes, Verrucomicrobia, and Chloroflexi, highlighting their ecological importance. Our study helps define the role of several poorly understood, uncultivated microbial groups in the turnover of benthic carbon derived from "dark" primary production.IMPORTANCE Little is known about the ecological role of uncultivated microbial populations in carbon turnover in benthic environments. To better understand this, we used quantitative stable isotope probing (qSIP) to quantify the abundance of diverse, specific groups of uncultivated bacteria and archaea involved in autotrophy and heterotrophy in a benthic lacustrine habitat. Our results provide quantitative evidence for active heterotrophic and autotrophic metabolism of several poorly understood microbial groups, thus demonstrating their relevance for carbon turnover in benthic settings. Archaeal ammonia oxidizers were significant drivers of in situ "dark" primary production supporting the growth of heterotrophic bacteria. These findings expand our understanding of the microbial populations within benthic food webs and the role of uncultivated microbes in benthic carbon turnover.

Project description: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.

Project description:The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H218O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation. IMPORTANCE Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.

Project description: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.

Project description:Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to (15)[Formula: see text] and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were (15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis-including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)-were all observed to be actively synthesized ((15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of (15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.

Dataset Information

Quantitative microbial ecology through stable isotope probing.

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Quantitative microbial ecology through stable isotope probing.

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