A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries

Authors

  • Gordon Webster,

    Corresponding author
    1. Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3TL, UK.
    2. School of Earth, Ocean and Planetary Sciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3YE, UK.
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  • Lynsey C. Watt,

    1. Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queens Road, Bristol, England BS8 1RJ, UK.
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  • Joachim Rinna,

    1. School of Earth, Ocean and Planetary Sciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3YE, UK.
    2. Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queens Road, Bristol, England BS8 1RJ, UK.
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  • John C. Fry,

    1. Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3TL, UK.
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  • Richard P. Evershed,

    1. School of Chemistry, University of Bristol, Cantocks Close, Bristol, England BS8 1TS, UK.
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  • R. John Parkes,

    1. School of Earth, Ocean and Planetary Sciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3YE, UK.
    2. Department of Earth Sciences, University of Bristol, Wills Memorial Building, Queens Road, Bristol, England BS8 1RJ, UK.
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  • Andrew J. Weightman

    1. Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3TL, UK.
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*E-mail websterg@cardiff.ac.uk; Tel. (+44) 2920874928; Fax (+44) 2920874326.

Summary

Marine sediment slurries enriched for anaerobic, sulfate-reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable-isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13C-labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 µM) were used over a 7-day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13C-glucose slurry but this changed with time, suggesting the presence of an active glucose-utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose-utilizers was unclear as 13C-enriched PLFA were common (16:0, 16:1, 18:1ω7, highest incorporation) and there was little difference between 12C- and 13C-DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13C-incorporation (∼10 times) into PLFA compared with 13C-acetate or 13C-pyruvate. 13C-PLFA in the 13C-acetate and 13C-pyruvate slurries were similar to each other and to those that developed in the 13C-glucose slurry after 4 days. These were more diagnostic, with branched odd-chain fatty acids (i15:0, a15:0 and 15:1ω6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate-reducing bacteria (SRB) and sequences related to these SRB were in the 13C-acetate-DNA dsrA gene library. The 13C-acetate-DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate-utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13C-DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13C-acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13C-DNA from the 13C-glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate-reducing conditions. Here we demonstrate that PLFA- and DNA-SIP can be used together in a sedimentary system, with low concentrations of 13C-substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.

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