scholarly journals Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density

2007 ◽  
Vol 73 (10) ◽  
pp. 3189-3195 ◽  
Author(s):  
Daniel H. Buckley ◽  
Varisa Huangyutitham ◽  
Shi-Fang Hsu ◽  
Tyrrell A. Nelson
Keyword(s):  
Stable Isotope ◽  
Natural Variation ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Density Gradients ◽  
Isotope Enrichment ◽  
Labeled Compounds ◽  
Contrast Variation ◽  
Functional Capabilities ◽  
Dna Density

ABSTRACT Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.

scholarly journals Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Ella T. Sieradzki ◽  
Benjamin J. Koch ◽  
Alex Greenlon ◽  
Rohan Sachdeva ◽  
Rex R. Malmstrom ◽  
...  
Keyword(s):  
Measurement Error ◽  
Experimental Design ◽  
Stable Isotope ◽  
Statistical Power ◽  
Quantitative Estimation ◽  
Cost Benefit ◽  
Data Interpretation ◽  
Stable Isotope Probing ◽  
Isotope Enrichment ◽  
Community Interactions

ABSTRACT 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.

scholarly journals DNA buoyant density shifts during 15N-DNA stable isotope probing

2007 ◽  
Vol 162 (4) ◽  
pp. 328-334 ◽  
Author(s):  
Alison M. Cupples ◽  
Elizabeth A. Shaffer ◽  
Joanne C. Chee-Sanford ◽  
Gerald K. Sims
Keyword(s):  
Stable Isotope ◽  
Buoyant Density ◽  
Stable Isotope Probing

scholarly journals Quantitative Microbial Ecology through Stable Isotope Probing

2015 ◽  
Vol 81 (21) ◽  
pp. 7570-7581 ◽  
Author(s):  
Bruce A. Hungate ◽  
Rebecca L. Mau ◽  
Egbert Schwartz ◽  
J. Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements ◽  
Isopycnic Centrifugation

ABSTRACTBacteria 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 tostableisotopeprobing (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 in18O and13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of18O into DNA from [18O]water. However, the increase in18O 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.

scholarly journals Quantitative microbial ecology through stable isotope probing

2015 ◽  
Author(s):  
Bruce A Hungate ◽  
Rebecca L Mau ◽  
Egbert Schwartz ◽  
J Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Glucose Addition ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements

Bacteria grow and transform elements at different rates, 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 determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. 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 non-labeled 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 demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in 18O and 13C composition after exposure to 18O-H2O or 13C-glucose. Addition of glucose increased assimilation of 18O into DNA from 18O-H2O. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

scholarly journals RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

2014 ◽  
Vol 80 (7) ◽  
pp. 2240-2247 ◽  
Author(s):  
Gerald W. Tannock ◽  
Blair Lawley ◽  
Karen Munro ◽  
Ian M. Sims ◽  
Julian Lee ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Bacterial Species ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Cells ◽  
Bacterial Populations ◽  
Content Type ◽  
And Function

ABSTRACTKnowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed thatBacteroides uniformis,Blautia glucerasea,Clostridium indolis, andBifidobacterium animaliswere the main users of the13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.B. uniformisutilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.

scholarly journals High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

2011 ◽  
Vol 78 (1) ◽  
pp. 163-169 ◽  
Author(s):  
Peter Andeer ◽  
Stuart E. Strand ◽  
David A. Stahl
Keyword(s):  
Stable Isotope ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Microbial Populations ◽  
Bacterial Isolates ◽  
Content Type ◽  
Restriction Fragment Length

ABSTRACTStable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)–TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli,Rhodococcus,Variovorax, andMicrobacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

scholarly journals Development of a Fatty Acid and RNA Stable Isotope Probing-Based Method for Tracking Protist Grazing on Bacteria in Wastewater

2010 ◽  
Vol 76 (24) ◽  
pp. 8222-8230 ◽  
Author(s):  
Steffen Kuppardt ◽  
Antonis Chatzinotas ◽  
Matthias Kästner
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cell Division ◽  
Stable Isotope ◽  
Pathogenic Bacteria ◽  
Buoyant Density ◽  
Stable Isotope Probing ◽  
Estimation Model ◽  
Large Shift ◽  
Actual Contribution

ABSTRACT Removal of potential pathogenic bacteria, for example, during wastewater treatment, is effected by sorption, filtration, natural die-off, lysis by viruses, and grazing by protists, but the actual contribution of grazing has never been assessed quantitatively. A methodical approach for analyzing the grazing of protists on 13C-labeled prey bacteria was developed which enables mass balances of the carbon turnover to be drawn, including yield estimation. Model experiments for validating the approach were performed in closed microcosms with the ciliate Uronema sp. and 13C-labeled Escherichia coli as model prey. The transfer of bacterial 13C into grazing protist biomass was investigated by fatty acid (FA) and RNA stable isotope probing (SIP). Uronema sp. showed ingestion rates of ∼390 bacteria protist−1 h−1, and the temporal patterns of 13C assimilation from the prey bacteria to the protist FA were identified. Nine fatty acids specific for Uronema sp. were found (20:0, i20:0, 22:0, 24:0, 20:1ω9c, 20:1ω9t, 22:1ω9c, 22:1ω9t, and 24:1). Four of these fatty acids (22:0, 20:1ω9t, 22:1ω9c, and 22:1ω9t) were enriched very rapidly after 3 h, indicating grazing on bacteria without concomitant cell division. Other fatty acids (20:0, i20:0, and 20:1ω9c) were found to be indicative of growth with cell division. The fatty acids were found to be labeled with a percentage of labeled carbon (atoms percent [atom%]) up to 50. Eighteen percent of the E. coli-derived 13C was incorporated into Uronema biomass, whereas 11% was mineralized. Around 5 mol bacterial carbon was necessary in order to produce 1 mol protist carbon (yx / s ≈ 0.2), and the temporal pattern of 13C labeling of protist rRNA was also shown. A consumption of around 1,000 prey bacteria (∼98 atom% 13C) per protist cell appears to be sufficient to provide detectable amounts of label in the protist RNA. The large shift in the buoyant density fraction of 13C-labeled protist RNA demonstrated a high incorporation of 13C, and reverse transcription-real-time PCR (RT-qPCR) confirmed that protist rRNA increasingly dominated in the heavy RNA fraction.

scholarly journals Quantitative microbial ecology through stable isotope probing

2015 ◽  
Author(s):  
Bruce A Hungate ◽  
Rebecca L Mau ◽  
Egbert Schwartz ◽  
J Gregory Caporaso ◽  
Paul Dijkstra ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Probing ◽  
Taxonomic Resolution ◽  
Isotope Enrichment ◽  
Glucose Addition ◽  
Density Fractions ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Quantitative Measurements

Bacteria grow and transform elements at different rates, 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 determining the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers. 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 non-labeled 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 demonstrate qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variation in 18O and 13C composition after exposure to 18O-H2O or 13C-glucose. Addition of glucose increased assimilation of 18O into DNA from 18O-H2O. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because glucose addition indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

scholarly journals Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments

PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0169554 ◽  
Author(s):  
Jingfeng Gao ◽  
Kailing Pan ◽  
Hongyu Li ◽  
Xiaoyan Fan ◽  
Lixin Sun ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Cesium Chloride ◽  
Stable Isotope Probing ◽  
Density Gradients
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