RNA stable isotope probing, a novel means of linking microbial community function to phylogeny.
Ontology highlight
ABSTRACT:
SUBMITTER: Manefield M
PROVIDER: S-EPMC129944 | biostudies-literature | 2002 Nov
REPOSITORIES: biostudies-literature
Publications
RNA stable isotope probing, a novel means of linking microbial community function to phylogeny.
Manefield Mike M Whiteley Andrew S AS Griffiths Robert I RI Bailey Mark J MJ
Applied and environmental microbiology 20021101 11
Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced wa ...[more]
Similar Datasets
Project description:We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine (13)C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotope-labeled [(13)C]glucose, [(13)C]propionate, or [(13)C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that (13)C labeling can be much lower, to the extent that delta(13)C ratios can be studied even at their natural abundances.
| S-EPMC2725495 | biostudies-literature
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.
| S-EPMC6122004 | biostudies-literature
Project description: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.
| S-EPMC4592864 | biostudies-literature
Project description:Growing bacteria have a high concentration of ribosomes to ensure sufficient protein synthesis, which is necessary for genome replication and cellular division. To elucidate whether metabolic activity of soil microorganisms is coupled with growth, we investigated the relationship between rRNA and DNA synthesis in a soil bacterial community using quantitative stable isotope probing (qSIP) with H218O. Most soil bacterial taxa were metabolically active and grew, and there was no significant difference between the isotopic composition of DNA and RNA extracted from soil incubated with H218O. The positive correlation between 18O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity.IMPORTANCE Using quantitative stable isotope probing of microbial RNA and DNA with H218O, we show that most soil taxa are metabolically active and grow because their nucleic acids are significantly labeled with 18O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities.
| S-EPMC5881069 | biostudies-literature
Project description:Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.
| S-EPMC3273032 | biostudies-literature
Project description:Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and 13C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.
| S-EPMC7852690 | biostudies-literature
Project description:The rate at which microorganisms grow and reproduce is fundamental to our understanding of microbial physiology and ecology. While soil microbiologists routinely quantify soil microbial biomass levels and the growth rates of individual taxa in culture, there is a limited understanding of how quickly microbes actually grow in soil. For this work, we posed the simple question: what are the growth rates of soil microorganisms? In this study, we measure these rates in three distinct soil environments using hydrogen-stable isotope probing of lipids with 2H-enriched water. This technique provides a taxa-agnostic quantification of in situ microbial growth from the degree of 2H enrichment of intact polar lipid compounds ascribed to bacteria and fungi. We find that growth rates in soil are quite slow and correspond to average generation times of 14 to 45 d but are also highly variable at the compound-specific level (4 to 402 d), suggesting differential growth rates among community subsets. We observe that low-biomass microbial communities exhibit more rapid growth rates than high-biomass communities, highlighting that biomass quantity alone does not predict microbial productivity in soil. Furthermore, within a given soil, the rates at which specific lipids are being synthesized do not relate to their quantity, suggesting a general decoupling of microbial abundance and growth in soil microbiomes. More generally, we demonstrate the utility of lipid-stable isotope probing for measuring microbial growth rates in soil and highlight the importance of measuring growth rates to complement more standard analyses of soil microbial communities.
| S-EPMC10120080 | biostudies-literature
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.
| S-EPMC4850331 | biostudies-literature
Project description:The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.
| S-EPMC1317343 | biostudies-literature
Project description:The goal of this field study was to provide insight into three distinct populations of microorganisms involved in in situ metabolism of phenol. Our approach measured 13CO2 respired from [13C]phenol and stable isotope probing (SIP) of soil DNA at an agricultural field site. Traditionally, SIP-based investigations have been subject to the uncertainties posed by carbon cross-feeding. By altering our field-based, substrate-dosing methodologies, experiments were designed to look beyond primary degraders to detect trophically related populations in the food chain. Using gas chromatography-mass spectrometry (GC/MS), it was shown that (13)C-labeled biomass, derived from primary phenol degraders in soil, was a suitable growth substrate for other members of the soil microbial community. Next, three dosing regimes were designed to examine active members of the microbial community involved in phenol metabolism in situ: (i) 1 dose of [13C]phenol, (ii) 11 daily doses of unlabeled phenol followed by 1 dose of [13C]phenol, and (iii) 12 daily doses of [13C]phenol. GC/MS analysis demonstrated that prior exposure to phenol boosted 13CO2 evolution by a factor of 10. Furthermore, imaging of 13C-treated soil using secondary ion mass spectrometry (SIMS) verified that individual bacteria incorporated 13C into their biomass. PCR amplification and 16S rRNA gene sequencing of 13C-labeled soil DNA from the 3 dosing regimes revealed three distinct clone libraries: (i) unenriched, primary phenol degraders were most diverse, consisting of alpha-, beta-, and gamma-proteobacteria and high-G+C-content gram-positive bacteria, (ii) enriched primary phenol degraders were dominated by members of the genera Kocuria and Staphylococcus, and (iii) trophically related (carbon cross-feeders) were dominated by members of the genus Pseudomonas. These data show that SIP has the potential to document population shifts caused by substrate preexposure and to follow the flow of carbon through terrestrial microbial food chains.
| S-EPMC1317415 | biostudies-literature