163 research outputs found

    Large D/H variations in bacterial lipids reflect central metabolic pathways

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    Large hydrogen-isotopic (D/H) fractionations between lipids and growth water have been observed in most organisms studied to date. These fractionations are generally attributed to isotope effects in the biosynthesis of lipids, and are frequently assumed to be approximately constant for the purpose of reconstructing climatic variables. Here, we report D/H fractionations between lipids and water in 4 cultured members of the phylum Proteobacteria, and show that they can vary by up to 500‰ in a single organism. The variation cannot be attributed to lipid biosynthesis as there is no significant change in these pathways between cultures, nor can it be attributed to changing substrate D/H ratios. More importantly, lipid/water D/H fractionations vary systematically with metabolism: chemoautotrophic growth (approximately −200 to −400‰), photoautotrophic growth (−150 to −250‰), heterotrophic growth on sugars (0 to −150‰), and heterotrophic growth on TCA-cycle precursors and intermediates (−50 to +200‰) all yield different fractionations. We hypothesize that the D/H ratios of lipids are controlled largely by those of NADPH used for biosynthesis, rather than by isotope effects within the lipid biosynthetic pathway itself. Our results suggest that different central metabolic pathways yield NADPH—and indirectly lipids—with characteristic isotopic compositions. If so, lipid δD values could become an important biogeochemical tool for linking lipids to energy metabolism, and would yield information that is highly complementary to that provided by ^(13)C about pathways of carbon fixation

    Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

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    Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with ^(13)C-labeled methane, ethane, or propane, we confirmed the incorporation of ^(13)C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in ^(13)C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, ^(13)C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, ^(13)C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the ^(13)C-labeled DNA may encode an ethane monooxygenase. Third, ^(13)C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes

    Compound-Specific δ^(34)S Analysis of Volatile Organics by Coupled GC/Multicollector-ICPMS

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    We have developed a highly sensitive and robust method for the analysis of δ^(34)S in individual organic compounds by coupled gas chromatography (GC) and multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). The system requires minimal alteration of commercial hardware and is amenable to virtually all sample introduction methods. Isobaric interference from O_2^+ is minimized by employing dry plasma conditions and is cleanly resolved at all masses using medium resolution on the Thermo Neptune MC-ICPMS. Correction for mass bias is accomplished using standard−sample bracketing with peaks of SF6 reference gas. The precision of measured δ^(34)S values approaches 0.1‰ for analytes containing >40 pmol S and is better than 0.5‰ for those containing as little as 6 pmol S. This is within a factor of 2 of theoretical shot-noise limits. External accuracy is better than 0.3‰. Integrating only the center of chromatographic peaks, rather than the entire peak, offers significant gain in precision and chromatographic resolution with minimal effect on accuracy but requires further study for verification as a routine method. Coelution of organic compounds that do not contain S can cause degraded analytical precision. Analyses of crude oil samples show wide variability in δ^(34)S and demonstrate the robustness and precision of the method in complex environmental samples

    Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria.

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    Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen-protium and deuterium-that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ(2)H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ(2)H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ(2)H suggest much potential as an environmental recorder of metabolism

    John M. Hayes 1940-2017. Father of isotopes in modern and ancient biogeochemical processes, biosynthetic carbon and hydrogen isotope fractionation and compound specific isotope analytical techniques

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    John Michael Hayes, Professor of chemistry and geology for 26 years at Indiana University (Bloomington) until 1996, then director of the National Ocean Sciences Accelerator Mass Spectrometry facility at Woods Hole Oceanographic Institution and adjunct professor at Harvard University until 2007, died peacefully at his home in Berkeley, California, on February 3rd, 2017

    Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry

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    Rationale: Sulfur isotope analysis of organic sulfur‐containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural‐abundance sulfur isotopic compositions of the sulfur‐containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields. Methods: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed‐phase high‐performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ³⁴S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature‐ramped chromatographic column and programmable helium carrier flow rates. Results: On‐column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1–0.3‰ 1 s.d.) δ³⁴S measurements of 1 to 10 μg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof‐of‐concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ³⁴S values of cysteine and methionine that can be connected to biosynthetic pathways. Conclusions: We have developed a sensitive, precise method for measuring the natural‐abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis

    Measurement of intact methane isotopologues, including ^(13)CH_3D

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    Methane (CH_4) is both a significant greenhouse gas and resource. Its present and past cycling can be studied through measurements of concentration and/or bulk isotopic ratios (^(13)C/^(12)C, D/H, and ^(14)C/^(12)C). Currently, isotope ratios are measured by mass spectrometric analysis of H_2 and CO_2 produced from CH_4, or by spectroscopy of CH_4. However, the interpretation of bulk isotopic variations of CH_4 are often equivocal, necessitating additional tracers
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