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Article
Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with H2 18O
Microbiology Open (2015)
  • Volker Brozel, South Dakota State University
Abstract

Current limitations in culture-based methods have lead to a reliance on culture-independent approaches, based principally on the comparative analysis of primary semantides such as ribosomal gene sequences.  DNA is remarkably stable in some environments, so its presence does not indicate live bacteria, but extracted ribosomal RNA (rRNA) has been viewed as an indicator of active cells.  Stable isotope probing (SIP) involves the incorporation of heavy isotopes into newly synthesized nucleic acids, and can be used to separate newly synthesized from existing DNA or rRNA.  H218O is currently the only potential universal bacterial substrate suitable for SIP of entire bacterial communities.  The aim of our work was to compare soil bacterial community composition as revealed by total versus SIP-labeled DNA and rRNA.  Soil was supplemented with H218O and after 38 days the DNA and RNA were co-extracted.   Heavy nucleic acids were separated out by CsCl and CsTFA density centrifugation.  The 16S rRNA gene pools were characterized by DGGE and pyrosequencing, and the sequence results analyzed using mothur.  The analysis indicated that total and active members of the same type of nucleic acid represented similar community structures, which suggested that most dominant OTUs in the total nucleic acid extracts contained active members.  It also supported that H218O was an effective universal label for stable isotope probing for both DNA and RNA.  DNA and RNA-derived diversity was dissimilar.  RNA more comprehensively recovered bacterial diversity than DNA because the most abundant OTUs were less numerous in RNA than DNA derived community data, and dominant OTU pools didn’t mask rare OTUs as much in RNA.  Because community composition derived from labeled and unlabeled (total) nucleic acids was so similar, stable isotope probing may not be necessary when attempting to characterize active soil microbial communities. 
Keywords
  • Bacterial diversity,
  • DNA,
  • H2 18O,
  • RNA,
  • SIP,
  • soil.
Disciplines
Publication Date
2015
DOI
doi: 10.1002/mbo3.230
Citation Information
Volker Brozel. "Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with H2 18O" Microbiology Open Vol. 4 Iss. 2 (2015) p. 208 - 219
Available at: http://works.bepress.com/volker-brozel/5/