A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.
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 microM) 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, 18:1omega7, 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 (approximately 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 15:1omega6) 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.
Webster
Gordon
G
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3TL, UK. websterg@cardiff.ac.uk
Watt
Lynsey C
LC
Rinna
Joachim
J
Fry
John C
JC
Evershed
Richard P
RP
Parkes
R John
RJ
Weightman
Andrew J
AJ
D016172:DNA Fingerprinting / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification; D005227:Fatty Acids; D005798:Genes, Bacterial / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology*; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013431:Sulfates / Q000378:metabolism; D016999:Sulfur-Reducing Bacteria / Q000145:classification* / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D005227:Fatty Acids; D010743:Phospholipids; D012336:RNA, Ribosomal, 16S; D013431:Sulfates