Hydrogen isotopic fractionation in lipid biosynthesis by H2-consuming Desulfobacterium autotrophicum

Campbell, Brian; Li, Chao; Sessions, Alex L.; Valentine, David L.

doi:10.1016/j.gca.2009.02.034

Cited by 49 publications

(71 citation statements)

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“…The pattern is supported by previous culture studies of bacteria (19)(20)(21) and plants (11,33) and is consistent with field observations. For example, fatty acids with odd carbon-numbered chains are substantially D-enriched (by up to 100‰) relative to those with even-numbered chains in marine POM (17).…”

Section: Discussionsupporting

confidence: 90%

“…The corresponding value of ␣ l/w ranged from 0.64 to 0.73 for different fatty acids (Table S3). Growth on formate yielded nearly identical results, even though formate is a potential source of H. We infer that H on formate exchanges with water to preclude the transmission of substrate H to fatty acids, in accord with our understanding of formate metabolism (29) and the recent results of Campbell et al (20). If X w is unknown, as is the case for most heterotrophic growth conditions, then a unique solution for X w , ␣ l/w , and ␣ l/s is not possible (31).…”

Section: Growth On Different Substrates Leads To Varying Fractionatiosupporting

confidence: 85%

“…Hydrocarbons produced by the green alga Botryococcus braunii were depleted in D relative to growth water by 197 to 358‰ (16). Fatty acids from the aerobic methanotroph Methylococcus capsulatus (19) were depleted by 20 to 70‰, whereas those in the sulfatereducing chemoautotroph Desulfobacterium autotrophicum were depleted by 190 to 360‰ (20). The most strongly fractionating organism reported to date is an H 2 ϩ CO 2 using acetogen, Sporomusa sp.…”

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

Zhang¹,

Gillespie

Sessions

2009

Proc. Natl. Acad. Sci. U.S.A.

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195

268

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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 ␦D 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.fatty acids ͉ fractionation ͉ metabolism ͉ hydrogen isotopes T he hydrogen-isotopic composition ( 2 H/ 1 H or D/H ratio, commonly expressed as a ␦D value) of lipids is being explored by scientists with diverse interests, including the origins of natural products (1, 2), biogeochemical cycles (3), petroleum systems (4), and paleoclimate (5-7). Because the D/H ratios of lipids are generally conserved over Ϸ10 6 -year time scales (8), they are a potentially useful tracer of biogeochemical pathways and processes in the environment. Most research to date has focused on higher plants, in which environmental water is the sole source of external hydrogen and consequently provides primary control over the D/H ratio of biosynthesized lipids (9). Although ␦D values for plant lipids and environmental water are generally well correlated, they are also substantially offset from each other. The biochemical basis for this lipid/water fractionation is not well understood. It is generally assumed to arise from a combination of isotope effects during photosynthesis and the biosynthesis of lipids (9-12), and is often treated as approximately constant to reconstruct isotopic compositions of environmental water as a paleoclimate proxy.There is, however, mounting evidence that the net D/H fractionation between lipids and water can vary by up to 150‰ in plants, even in the same organism (12-16). Modest fractionations associated with fatty acid elongation and desaturation ...

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Section: Discussionsupporting

confidence: 90%

Section: Growth On Different Substrates Leads To Varying Fractionatiosupporting

confidence: 85%

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confidence: 99%

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

Zhang¹,

Gillespie

Sessions

2009

Proc. Natl. Acad. Sci. U.S.A.

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195

268

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“…This spectral pattern reflects a substitution of C-H X by C-D X in newly synthesized macromolecules in the presence of heavy water, and it is safe to assume that an important fraction of the D incorporation is found in lipids, which contribute roughly 25-30% to total cellular C-H (45, 46). Lipids from autotrophic and heterotrophic organisms show a predominance of water-derived protons (or D + in the presence of heavy water) (33,35,47) as protons from water are transferred to NAD(P) during its reduction, and these protons are then incorporated into lipids via the known fatty acid biosynthesis pathways (31-33). D will, of course, also be incorporated to some extent via other processes, such as amino acid and carbohydrate biosynthesis (37, 48).…”

Section: Resultsmentioning

confidence: 99%

Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells

Berry

Mader

Lee

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

368

512

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Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D 2 O) combined with Raman microspectroscopy. Incorporation of D 2 O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscaleresolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D 2 O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating the potential of the nondestructive D 2 O-Raman approach for targeted sorting of microbial cells with defined functional properties for singlecell genomics.ecophysiology | single-cell microbiology | carbohydrate utilization | nitrifier | Raman microspectroscopy M icroorganisms play a vital role in many environments. They mediate global biogeochemical cycles, catalyze biotechnological processes, and contribute to health and disease in the human body. The in situ study of microbial activity in natural and engineered ecosystems is therefore of great interest. For this purpose, several elegant methods have been established that use either transcriptional or translational activity of community members (i.e., metatranscriptomics, metaproteomics) (1-3) or the incorporation of isotopically labeled substrates into biomolecules (4-10) to infer the ecophysiology of microbes in such systems. However, these bulk techniques do not offer sufficient spatial resolution to study microbial activities at the micrometer scale. Therefore, important information can be overlooked because microbial communities are frequently spatially structured (e.g., biofilms) (11) and contain populations with life cycles (12,13). Furthermore, even apparently identical cells in clonal populations can have strongly divergent activities (14).Consequently, microbial ecophysiology is ideally studied also at the level of the single cell, but only a restricted number of approaches exist for determining physiological properties of individual cells in a microbial community. For exa...

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“…Mid-ocean ridge hydrothermal systems Crist and Dalin, 1933;Gould et al, 1934;Hall et al, 1934;Koepp, 1978;Lyon and Hulston, 1984;Lécluse and Robert, 1994;Campbell et al, 2009; …”

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The geochemistry of methane isotopologues

Wang¹

2017

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This thesis documents the origin, distribution, and fate of methane and several of its isotopic forms on Earth. Using observational, experimental, and theoretical approaches, I illustrate how the relative abundances of 12 CH 4 , 13 CH 4 , 12 CH 3 D, and 13 CH 3 D record the formation, transport, and breakdown of methane in selected settings.Chapter 2 reports precise determinations of 13 CH 3 D, a "clumped" isotopologue of methane, in samples collected from various settings representing many of the major sources and reservoirs of methane on Earth. The results show that the information encoded by the abundance of 13 CH 3 D enables differentiation of methane generated by microbial, thermogenic, and abiogenic processes. A strong correlation between clumped-and hydrogen-isotope signatures in microbial methane is identified and quantitatively linked to the availability of H 2 and the reversibility of microbially-mediated methanogenesis in the environment. Determination of 13 CH 3 D in combination with hydrogen-isotope ratios of methane and water provides a sensitive indicator of the extent of C-H bond equilibration, enables fingerprinting of methane-generating mechanisms, and in some cases, supplies direct constraints for locating the waters from which migrated gases were sourced. Chapter 3 applies this concept to constrain the origin of methane in hydrothermal fluids from sediment-poor vent fields hosted in mafic and ultramafic rocks on slow-and ultraslow-spreading mid-ocean ridges. The data support a hypogene model whereby methane forms abiotically within plutonic rocks of the oceanic crust at temperatures above ca. 300• C during respeciation of magmatic volatiles, and is subsequently extracted during active, convective hydrothermal circulation. Chapter 4 presents the results of culture experiments in which methane is oxidized in the presence of O 2 by the bacterium Methylococcus capsulatus strain Bath. The results show that the clumped isotopologue abundances of partially-oxidized methane can be predicted from knowledge of 13 C/ 12 C and D/H isotope fractionation factors alone.: Shuhei Ono, Ph.D. Acknowledgments I fear that this section will not do justice to the people I have not the space to thank individually. Alas, here goes. To those whose names are not specifically listed here, thank you too. I wish first to thank Shuhei Ono. The work this thesis represents could not have happened without his bold vision and the license to explore and question that working in his lab afforded. His unbridled optimism, breadth of scientific curiosity, personal integrity, and accessibility to his personnel are traits I one day hope to emulate. I thank him for his nonjudgmental yet appropriately measured support of many of my ideas-even those that led nowhere-, for believing in me even when I found it difficult to do so myself, and for teaching me to change pump oil, build vacuum lines, and drink Icelandic vodka.I also wish to thank my thesis committee members Jeff Seewald, Roger Summons, and John Pohlman, for their in...

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Hydrogen isotopic fractionation in lipid biosynthesis by H2-consuming Desulfobacterium autotrophicum

Cited by 49 publications

References 50 publications

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

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

Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells

The geochemistry of methane isotopologues

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Hydrogen isotopic fractionation in lipid biosynthesis by H2-consuming Desulfobacterium autotrophicum

Cited by 49 publications

References 50 publications

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

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

Tracking heavy water (D 2 O) incorporation for identifying and sorting active microbial cells

The geochemistry of methane isotopologues

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Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells