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 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 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 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.

Calculation of substrate flux using stable isotopes

1988 ◽  
Vol 254 (4) ◽  
pp. E526-E531 ◽  
Author(s):  
J. Rosenblatt ◽  
R. R. Wolfe
Keyword(s):  
Steady State ◽  
Stable Isotope ◽  
Radioactive Isotopes ◽  
Natural Occurrence ◽  
Isotopic Enrichment ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Substrate Kinetics ◽  
Made In

The use of stable isotope tracers to calculate substrate kinetics in humans is favored over the use of radioactive isotopes because of their greater safety and versatility. However, potential complications not met when dealing with radioactive tracers are caused by 1) the natural occurrence of the stable isotope used as a tracer and 2) the necessity to administer the tracer in an amount that cannot be treated as "massless." We therefore found it desirable to derive a theoretically valid equation for calculating the rate of appearance, Ra, of a substrate under steady-state conditions using a stable isotope tracer. This theoretically valid equation yields results that differ from those of the equations conventionally used to calculate (endogenous) Ra in steady state. Quantitative determination of the error in one of these equations revealed that for tracers commonly used in metabolic studies the error is negligible, whereas the error made in the other equation is likely to be quite high in commonly encountered situations. Finally, to allow for proper use of different definitions of isotopic enrichment that have arisen from practical considerations, we use the results derived above to determine valid equations for Ra appropriate to the two prevalent definitions.

scholarly journals Identification of Acetate-Assimilating Microorganisms under Methanogenic Conditions in Anoxic Rice Field Soil by Comparative Stable Isotope Probing of RNA

2006 ◽  
Vol 73 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Tomoyuki Hori ◽  
Matthias Noll ◽  
Yasuo Igarashi ◽  
Michael W. Friedrich ◽  
Ralf Conrad
Keyword(s):  
Stable Isotope ◽  
Rice Field ◽  
Ferric Iron ◽  
Stable Isotope Probing ◽  
Polymorphism Analysis ◽  
Field Soil ◽  
Bacterial Populations ◽  
Density Fractions ◽  
Reverse Transcription Pcr ◽  
Rice Field Soil

ABSTRACT Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.

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

2020 ◽  
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 ◽  
Stable Isotope Probing ◽  
Community Interactions ◽  
Density Fractions ◽  
And Function

AbstractQuantitative stable isotope probing (qSIP) estimates the degree of incorporation of an isotope tracer into nucleic acids of metabolically active organisms and can be applied to microorganisms growing in complex communities, such as the microbiomes of soil or water. As such, qSIP has the potential to link microbial biodiversity and biogeochemistry. As with any technique involving quantitative estimation, qSIP involves measurement error; a more complete understanding of error, precision and statistical power will aid in the design of qSIP experiments and interpretation of qSIP data. We used several existing qSIP datasets of microbial communities found in soil and water to evaluate how variance in the estimate of isotope incorporation depends on organism abundance and on the resolution of the density fractionation scheme. We also assessed statistical power for replicated qSIP studies, and sensitivity and specificity for unreplicated designs. We found that variance declines as taxon abundance increases. Increasing the number of density fractions reduces variance, although the benefit of added fractions declines as the number of fractions increases. Specifically, nine fractions appear to be a reasonable tradeoff between cost and precision for most qSIP applications. Increasing replication improves power and reduces the minimum detectable threshold for inferring isotope uptake to 5 atom%. Finally, we provide evidence for the importance of internal standards to calibrate the %GC to mean weighted density regression per sample. These results should benefit those designing future SIP experiments, and provide a reference for metagenomic SIP applications where financial and computational limitations constrain experimental scope.ImportanceOne 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 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 fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and function 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 replication, and cost-benefit balanced scenarios for SIP experimental design.

scholarly journals Use of stable isotopes to study carbohydrate and fat metabolism at the whole-body level

1999 ◽  
Vol 58 (4) ◽  
pp. 953-961 ◽  
Author(s):  
Andrew R. Coggan
Keyword(s):  
Stable Isotope ◽  
Radioactive Isotope ◽  
Fat Metabolism ◽  
Whole Body ◽  
Selected Ion Monitoring ◽  
Stable Isotope Tracers ◽  
Isotope Tracers ◽  
Isotope Tracer ◽  
Body Level ◽  
Muscle Biopsies

The present review discusses the advantages and limitations of using stable-isotope tracers to assess carbohydrate and fat metabolism at the whole-body level. One advantage of stable-(v. radioactive-) isotope tracers is the relative ease with which the location of a label within a molecule can be determined using selected-ion-monitoring GC-mass spectrometry (SIM-GC- MS). This technique minimizes potential problems due to label recycling, allows the use of multiple-labelled compounds simultaneously (e.g. to quantify glucose cycling), and perhaps most importantly, has led to the development of unique stable-isotope methods for, for example, quantifying gluconeogenesis. However, the limited sensitivity of SIM-GC-MS sometimes requires that relatively large amounts of a stable-isotope tracer be used, thus increasing cost and potentially altering metabolism. At least theoretically, stable- (or radioactive-) isotope tracers can also be used in conjunction with indirect calorimetry to estimate utilization of muscle glycogen or triacylglycerol stores, thus potentially circumventing the need to obtain muscle biopsies. These calculations, however, require certain critical assumptions, which if incorrect could lead to major errors in the values obtained. Despite such limitations, stable-isotope tracers provide a powerful and sometimes unique tool for investigating carbohydrate and fat metabolism at the whole-body level. With continuing advances in availability, instrumentation and methods, it is likely that stable-isotope tracers will become increasingly important in the immediate future.

scholarly journals Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover

2019 ◽  
Vol 219 (1) ◽  
Author(s):  
Derek P. Narendra ◽  
Christelle Guillermier ◽  
Frank Gyngard ◽  
Xiaoping Huang ◽  
Michael E. Ward ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Cell Biology ◽  
Isotope Labeling ◽  
Imaging Mass Spectrometry ◽  
Lateral Resolution ◽  
Dynamic State ◽  
Metabolic Labeling ◽  
Isotope Tracers ◽  
Lysosomal Protein

Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.

Calculation of substrate turnover rate in stable isotope tracer studies

1983 ◽  
Vol 245 (3) ◽  
pp. E308-E311 ◽  
Author(s):  
K. Y. Tserng ◽  
S. C. Kalhan
Keyword(s):  
Stable Isotope ◽  
Turnover Rate ◽  
Gas Chromatography Mass Spectrometry ◽  
Isotope Enrichment ◽  
Standard Curve ◽  
Isotope Tracer ◽  
Steady State Kinetics ◽  
Constant Rate ◽  
Peak Abundance

The determination of substrate turnover rate with stable isotope-labeled compounds has advantages of being safe and applicable in the study of children and pregnant women. Currently, a majority of these studies has been performed with primed constant-rate infusion technique. The isotope enrichment of the substrate in the plasma is measured by gas chromatography-mass spectrometry (GCMS). The turnover rate is then calculated from steady-state kinetics. A number of different equations have been used by various investigators for this purpose. Based on theoretical consideration and experimental data, it is concluded that the equation P = [(1/E) - 1] I or P = (y/x) I should be used for a correct turnover rate calculation, where P is turnover rate in mumol X kg-1, E the isotope enrichment, I the infusion rate in mumol X kg-1 X min-1, and y/x the mole ratio of tracee to tracers. A GCMS standard curve constructed from isotope enrichment versus isotope peak abundance should be used for the former equation, whereas a standard curve constructed from mole ratio (x/y) versus isotope ratio should be used for the latter equation. Interchange of standard curves or use of other equations will produce erroneous turnover rate. This is especially significant when a low enriched isotope-labeled compound is used as a tracer.

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