Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C₆ to C₁₂) and 1-¹³C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d₁₄-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-¹³C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that ¹³C nuclei from 1-¹³C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.     

Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds

Stable isotope fractionation was studied during the degradation of m-xylene, o-xylene, m-cresol, and p-cresol with two pure cultures of sulfate-reducing bacteria. Degradation of all four compounds is initiated by a fumarate addition reaction by a glycyl radical enzyme, analogous to the well-studied benzylsuccinate synthase reaction in toluene degradation. The extent of stable carbon isotope fractionation caused by these radical-type reactions was between enrichment factors () of −1.5 and −3.9‰, which is in the same order of magnitude as data provided before for anaerobic toluene degradation. Based on our results, an analysis of isotope fractionation should be applicable for the evaluation of in situ bioremediation of all contaminants degraded by glycyl radical enzyme mechanisms that are smaller than 14 carbon atoms. In order to compare carbon isotope fractionations upon the degradation of various substrates whose numbers of carbon atoms differ, intrinsic (_intrinsic) were calculated. A comparison of _intrinsic at the single carbon atoms of the molecule where the benzylsuccinate synthase reaction took place with compound-specific elucidated that both varied on average to the same extent. Despite variations during the degradation of different substrates, the range of found for glycyl radical reactions was reasonably narrow to propose that rough estimates of biodegradation in situ might be given by using an average if no fractionation factor is available for single compounds.     

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [¹³C₆]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.     

Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons

¹³C/¹²C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Stable Isotope Labeling in Zebrafish Allows in Vivo Monitoring of Cardiac Morphogenesis

Quantitative proteomics is an important tool to study biological processes, but so far it has been challenging to apply to zebrafish. Here, we describe a large scale quantitative analysis of the zebrafish proteome using a combination of stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS). Proteins derived from the fully labeled fish were used as a standard to quantify changes during embryonic heart development. LC-MS-assisted analysis of the proteome of activated leukocyte cell adhesion molecule zebrafish morphants revealed a down-regulation of components of the network required for cell adhesion and maintenance of cell shape as well as secondary changes due to arrest of cellular differentiation. Quantitative proteomics in zebrafish using the stable isotope-labeling technique provides an unprecedented resource to study developmental processes in zebrafish.Over the past years, mass spectrometry-based proteomics has been widely used to analyze complex biological samples (1). Although the latest generation of MS instrumentation allows proteome-wide analysis, protein quantitation is still a challenge (2, 3). Metabolic labeling using stable isotopes has been used for almost a century. Today, the most commonly used techniques for relative protein quantification are based on ¹⁵N labeling and stable isotope labeling by amino acids in cell culture (SILAC)¹ (4, 5). SILAC was initially developed for cell culture experiments, and recent approaches extended labeling to living organisms, including bacteria (6), yeast (7), flies (8), worms (9), and rodents (10, 11). In addition, several pulsed SILAC (also known as dynamic SILAC) experiments were performed to assess protein dynamics in cell culture and living animals (12–15).The zebrafish (Danio rerio) has proved to be an important model organism to study developmental processes. It also serves as a valuable tool to investigate basic pathogenic principles of human diseases such as cardiovascular disorders and tissue regeneration (16). So far, most researchers rely on immunohistochemistry and Western blots for semi-quantitative protein analysis, an approach that is hampered by the paucity of reliable antibodies in zebrafish. Proteomics approaches that depend on two-dimensional gel approaches (17–19) have not gained wide popularity because of issues with workload, reproducibility, and sensitivity (20, 21).Another approach for protein quantitation is the chemical modification of peptides, and so far several isobaric tagging methods, including ICAT (22), iTRAQ (23), ¹⁸O (24), and dimethyl labeling (25), have been proven to be successful methods.Recently, a quantitative phosphopeptide study based on dimethyl labeling in zebrafish showed the consequences of a morpholino-based kinase knockdown (26). However, each chemical modification bears the risk of nonspecific and incomplete labeling, which complicates mass spectrometric data interpretation.Alternatively, a metabolic labeling study with stable isotopes was recently performed on adult zebrafish by the administration of a mouse diet containing [¹³C₆]lysine (Lys-6) (27). Feeding adult zebrafish with the Lys-6-containing mouse chow leads to an incorporation rate of 40%, and SILAC labeling was used to investigate protein and tissue turnover.Here, we have developed a SILAC fish diet made in-house for the complete SILAC labeling of zebrafish. We established a Lys-6-containing diet as a universal fish food for larval and adult zebrafish. The method allows accurate quantitation of large numbers of proteins, and we proved our approach by the analysis of embryonic heart development. In addition, we investigated the consequences of the morpholino-based activated leukocyte cell adhesion molecule (ALCAM) knockdown during development and identified the lipid anchor protein Paralemmin as a down-regulated protein during heart development. Our approach yielded a huge resource of protein expression data for zebrafish development and provided the basis for more refined studies depending on accurate SILAC protein quantification.     

Stable Carbon Isotope Fractionation by Sulfate-Reducing Bacteria

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and δ¹³C values were determined for gaseous CO₂, organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO₂, and the cell biomass were small, ranging from 0 to 2‰. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9‰. SRB grown lithoautotrophically consumed less than 3% of the available CO₂ and exhibited substantial discrimination (calculated as isotope fractionation factors [α]), as follows: for Desulfobacterium autotrophicum, α values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the α value was 0.0138, and for Desulfotomaculum acetoxidans, the α value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO₂ resulted in biomass with a δ¹³C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H₂), ecological forces can also influence carbon isotope discrimination by SRB.     

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

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.     

Aberrant Water Homeostasis Detected by Stable Isotope Analysis

While isotopes are frequently used as tracers in investigations of disease physiology (i.e., ¹⁴C labeled glucose), few studies have examined the impact that disease, and disease-related alterations in metabolism, may have on stable isotope ratios at natural abundance levels. The isotopic composition of body water is heavily influenced by water metabolism and dietary patterns and may provide a platform for disease detection. By utilizing a model of streptozotocin (STZ)-induced diabetes as an index case of aberrant water homeostasis, we demonstrate that untreated diabetes mellitus results in distinct combinations, or signatures, of the hydrogen (δ²H) and oxygen (δ¹⁸O) isotope ratios in body water. Additionally, we show that the δ²H and δ¹⁸O values of body water are correlated with increased water flux, suggesting altered blood osmolality, due to hyperglycemia, as the mechanism behind this correlation. Further, we present a mathematical model describing the impact of water flux on the isotopic composition of body water and compare model predicted values with actual values. These data highlight the importance of factors such as water flux and energy expenditure on predictive models of body water and additionally provide a framework for using naturally occurring stable isotope ratios to monitor diseases that impact water homeostasis.     

Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic Archaea

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C₁ compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (−83‰ to −72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.     

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Utilization of a Stable Isotope of Oxygen by Pseudomonas fluorescens

Oxygen utilization is defined in this investigation as the terminal use of oxygen in respiration, i.e., the formation of water. A culture of Pseudomonas fluorescens was allowed to respire in an atmosphere of O¹⁸. The production of H₂O¹⁸ was measured during two test runs of 124 and 232 min each. During the first run, 0.505 mmole of H₂O¹⁸ was produced. The second run produced 0.460 mmole of H₂O¹⁸. H₂O¹⁸ production took place throughout the course of each of the runs.     

Dechlorination of Chlorocatechols by Stable Enrichment Cultures of Anaerobic Bacteria

Metabolically stable anaerobic cultures obtained by enrichment with 5-bromovanillin, 5-chlorovanillin, catechin, and phloroglucinol were used to study dechlorination of chlorocatechols. A high degree of specificity in dechlorination was observed, and some chlorocatechols were appreciably more resistant to dechlorination than others: only 3,5-dichlorocatechol, 4,5-dichlorocatechol, 3,4,5-trichlorocatechol, and tetrachlorocatechol were dechlorinated, and not all of them were dechlorinated by the same consortium. 3,5-Dichlorocatechol produced 3-chlorocatechol, 4,5-dichlorocatechol produced 4-chlorocatechol, and 3,4,5-trichlorocatechol produced either 3,5-dichlorocatechol or 3,4-dichlorocatechol; tetrachlorocatechol produced only 3,4,6-trichlorocatechol. Incubation of uncontaminated sediments without additional carbon sources brought about dechlorination of 3,4,5-trichlorocatechol to 3,5-dichlorocatechol. O-demethylation of chloroguaiacols was generally accomplished by enrichment cultures, except that catechin enrichment was unable to O-demethylate tetrachloroguaiacol. None of the enrichments dechlorinated any of the polychlorinated phenols examined. The results suggested that dechlorination was not dependent on enrichment with or growth at the expense of chlorinated compounds and that it would be premature to formulate general rules for the structural dependence of the dechlorination reaction.     

Transport of Magnesium by a Bacterial Nramp-Related Gene

Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5–2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.     

Transformations of Halogenated Aromatic Aldehydes by Metabolically Stable Anaerobic Enrichment Cultures

Metabolically stable enrichment cultures of anaerobic bacteria obtained by elective enrichment of sediment samples from the Baltic Sea and Gulf of Bothnia have been used to study the oxidation and reduction of the aldehyde group of various halogenated aromatic aldehydes. During the transformation of 5- and 6-chlorovanillin, 6-bromovanillin, 3-chloro-4-hydroxybenzaldehyde, 3,5-dichloro-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzaldehyde, it was shown that synthesis of the corresponding carboxylic acids, which were the principal metabolites, was invariably accompanied by partial reduction of the aldehyde to a hydroxymethyl group in yields of between 3 and 30%. Complete reduction to a methyl group was observed with some of the halogenated vanillins, but to an extremely limited extent with the halogenated 4-hydroxybenzaldehydes. One consortium produced both the hydroxymethyl and methyl compounds from both 5- and 6-chlorovanillin: it was therefore assumed that the methyl compound was the ultimate reduction product. On the basis of the kinetics of formation of the metabolites, it was concluded that the oxidation and reduction reactions were mechanistically related. In addition to these oxidations and reductions, dehalogenation was observed with one of the consortia. In contrast to the transformations of 5- and 6-chlorovanillin, which produced chlorinated methylcatechols, the corresponding compounds were not observed with 5- and 6-bromovanillin: the former was debrominated, forming 4-methylcatechol, whereas the latter produced 6-bromovanillyl alcohol without demethylation. Similarly, although 3-chloro-4-hydroxybenzaldehyde formed the chlorinated carboxylic acid and the benzyl alcohol, the 3-bromo compound was debrominated with formation of 4-hydroxybenzoic acid and, ultimately, phenol. On prolonged incubation, the halogenated carboxylic acids were generally decarboxylated, so that the final products from these substrates were halogenated catechols or phenols. Reductive processes of the type revealed in this study might therefore plausibly occur in the environment during anaerobic transformation of halogenated aromatic aldehydes containing hydroxyl and/or methoxyl groups.     

Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools

We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. Λ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bss-containing cell extracts (in vitro studies) were found to be similar to previously reported data from analogous experiments with whole cells (in vivo studies), proving that Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3- to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the α subunit of Bss, correlated with the observed differences in Λ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.     

Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.     

Quantification of the Dynamic Phosphorylation Process of ERK Using Stable Isotope Dilution Selective Reaction Monitoring Mass Spectrometry

Mitogen‐activated protein (MAP) kinase signaling is critical for various cellular responses, including cell proliferation, differentiation, and cell death. The MAP kinase cascade is conserved in the eukaryotic kingdom as a three‐tiered kinase module—MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase—that transduces signals via sequential phosphorylation upon stimulation. Dual phosphorylation of MAP kinase on the conserved threonine‐glutamic acid‐tyrosine (TEY) motif is essential for its catalytic activity and signal activation; however, the molecular mechanism by which the two residues are phosphorylated remains elusive. In the present study, the pattern of dual phosphorylation of extracellular signal‐regulated kinase (ERK) is profiled on the TEY motif using stable isotope dilution (SID)‐selective reaction monitoring (SRM) mass spectrometry (MS) to elucidate the order and magnitude of endogenous ERK phosphorylation in cellular model systems. The SID‐SRM‐MS analysis of phosphopeptides demonstrates that tyrosine phosphorylation in the TEY motif is dynamic, while threonine phosphorylation is static. Analyses of the mono‐phosphorylatable mutants ERK^T202A and ERK^Y204F indicate that phosphorylation of tyrosine is not affected by the phosphorylation state of threonine, while threonine phosphorylation depends on tyrosine phosphorylation. The data suggest that dual phosphorylation of ERK is a highly ordered and restricted mechanism determined by tyrosine phosphorylation.     

Stable Isotope Techniques and Applications for Primatologists

Stable isotope biogeochemistry is useful for quantifying the feeding ecology of modern and extinct primates. Over the past three decades, substantial advances have been made in our knowledge of the physiological causes of isotopic patterns as well as effective methodology to prepare samples for isotopic analysis. Despite these advances, the potential of stable isotope biogeochemistry has yet to be fully exploited by primate researchers, perhaps due to the prolific and somewhat daunting nature of the isotopic literature. I here aim to present a cogent overview of stable isotope applications to nonhuman primate feeding ecology. I review the factors that influence ecological patterns in carbon, nitrogen, and oxygen stable isotopes. I present methods for collecting and preparing samples of tooth enamel and bone mineral hydroxyapatite, bone collagen, fur and hair keratin, blood, feces, and urine for isotope analysis. I discuss both the existing and potential applications of these isotopic patterns to primate feeding ecology. Lastly, I point out some of the pitfalls to avoid when interpreting and comparing isotopic results.     

Quantitative Microbial Ecology through Stable Isotope Probing

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 ¹⁸O and ¹³C composition after exposure to [¹⁸O]water or [¹³C]glucose. The addition of glucose increased the assimilation of ¹⁸O into DNA from [¹⁸O]water. However, the increase in ¹⁸O 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.